GITR Antibodies And Methods Of Inducing Or Enhancing An Immune Response

ABSTRACT

The present invention provides binding molecules that specifically bind to GITR, e.g., human GITR (hGITR), on T cells and dendritic cells. Binding molecules of the invention are characterized by binding to hGITR with high affinity, in the presence of a stimulating agent, e.g., CD3, are agonistic, and abrogate the suppression of Teff cells by Treg cells. Various aspects of the invention relate to binding molecules, and pharmaceutical compositions thereof, as well as nucleic acids, recombinant expression vectors and host cells for making such binding molecules. Methods of using a binding molecule of the invention to detect human GITR or to modulate human GITR activity, either in vitro or in vivo, are also encompassed by the invention.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/345,182, filed Nov. 7, 2016, which is a continuation of U.S.application Ser. No. 14/665,533, filed Mar. 23, 2015, which issued asU.S. Pat. No. 9,493,572, and which is a continuation of U.S. applicationSer. No. 13/782,656, filed Mar. 1, 2013, which issued as U.S. Pat. No.9,028,823, and which is a continuation of U.S. application Ser. No.12/753,402, filed Apr. 2, 2010, which issued as U.S. Pat. No. 8,388,967,and which is a divisional of U.S. application Ser. No. 11/389,880, filedMar. 27, 2006, which issued as U.S. Pat. No. 7,812,135, and which claimsthe benefit of priority to U.S. Provisional Application No. 60/665,322,filed on Mar. 25, 2005, and U.S. Provisional Application No. 60/687,265,filed on Jun. 3, 2005. The entire teachings of each of the aboveapplications are herein incorporated by reference.

BACKGROUND OF THE INVENTION

Members of the tumor necrosis factor and TNF receptor (TNFR) superfamilyregulate diverse biologic functions, including cell proliferation,differentiation, and survival. Using differential display to identify Tcell mRNAs induced by the synthetic glucocorticoid hormonedexamethasone, Nocentini et al. ((1997) Proc. Natl. Acad. Sci., USA94:6216-6221997) identified a mouse cDNA encoding a novel member of theTNFR family. The corresponding gene was designated GITR forglucocorticoid-induced TNFR family-related gene (also known asTNFRSF18). Like other TNFRs, the predicted GITR protein containscysteine-rich repeats in the extracellular domain. In addition, theintracellular domain of GITR shares significant homology with those ofthe mouse and human TNFRs, 4-1BB and CD27. Nocentini et al. ((1997)Proc. Natl. Acad. Sci., USA 94:6216-6221997) demonstrated that the GITRgene is induced in T cells by dexamethasone as well as by othercell-activating stimuli. GITR expression protects T cells from apoptosisinduced by treatment with anti-CD3 antibodies, but not by otherapoptotic agents.

Shimizu et al. ((2002) Nat Immunol 3:135-42) found that GITR waspredominantly expressed on CD4+CD25+ regulatory T cells. However, GITRis also expressed on conventional CD4+ and CD8+ T cells, and itsexpression is enhanced rapidly after activation. In vitro studies haveshowed that GITR plays a key role in the peripheral tolerance that ismediated by these cells and abrogates the suppressive function ofCD4+CD25+ regulatory T cells (Shimizu et al. (2002) Nat Immunol3:135-42; McHugh et al. (2002) Immunity 16:311-23).

The development of agents useful in modulating signaling via GITR wouldbe of great benefit.

SUMMARY OF THE INVENTION

The present invention provides binding molecules that specifically bindto GITR, e.g., human GITR (hGITR), on cells, such as T cells anddendritic cells. The binding molecules of the invention arecharacterized by binding to hGITR with high affinity, are agonistic inthe presence of a stimulating agent, e.g., CD3, and abrogate thesuppression of T effector (Teff) cells by T regulatory (Treg) cells.

One aspect of the invention features a binding molecule comprising theamino acid sequence of SEQ ID NO.:1, optionally comprising a leadersequence.

In another aspect, the invention features a binding molecule comprisingthe amino acid sequence of SEQ ID NO.:66, optionally comprising a leadersequence.

In another aspect, the invention features a binding molecule comprisingthe amino acid sequence of SEQ ID NO.:2, optionally comprising a leadersequence.

Another aspect of the invention features a binding molecule comprisingthe amino acid sequence of SEQ ID NO:58, optionally comprising a leadersequence.

One aspect of the invention features a binding molecule comprising theamino acid sequence of SEQ ID NO.:59, optionally comprising a leadersequence.

In another aspect, the invention features a binding molecule comprisingthe amino acid sequence of SEQ ID NO.:60, optionally comprising a leadersequence.

In one aspect, the invention features a binding molecule comprising theamino acid sequence of SEQ ID NO.:61, optionally comprising a leadersequence.

In another aspect, the invention features a binding molecule comprisingthe amino acid sequence of SEQ ID NO.:62, optionally comprising a leadersequence.

One aspect of the invention features a binding molecule comprising theamino acid sequence of SEQ ID NO.:63, optionally comprising a leadersequence.

Yet another aspect of the invention features a binding moleculecomprising at least one complementarity determining region (CDR) aminoacid sequence selected from the group consisting of: SEQ ID NO.:3, SEQID NO.:4, or SEQ ID NO:19, and SEQ ID NO.:5. In one embodiment, thebinding molecule comprises at least two complementarity determiningregion (CDR) amino acid sequence selected from the group consisting of:SEQ ID NO.:3, SEQ ID NO.:4, or SEQ ID NO:19, and SEQ ID NO.:5. Inanother embodiment, the binding molecule comprises at least threecomplementarity determining region (CDR) amino acid sequence selectedfrom the group consisting of: SEQ ID NO.:3, SEQ ID NO.:4, or SEQ IDNO:19, and SEQ ID NO.:5.

Another aspect of the invention features a binding molecule comprisingat least one complementarity determining region (CDR) amino acidsequence selected from the group consisting of: SEQ ID NO.:6, SEQ IDNO.:7, and SEQ ID NO.:8. In one embodiment, the binding moleculecomprises at least two complementarity determining region (CDR) aminoacid sequence selected from the group consisting of: SEQ ID NO.:6, SEQID NO.:7, and SEQ ID NO.:8. In another embodiment, the binding moleculecomprises at least three complementarity determining region (CDR) aminoacid sequence selected from the group consisting of: SEQ ID NO.:6, SEQID NO.:7, and SEQ ID NO.:8.

Another aspect of the invention features a binding molecule comprisingthe CDRs shown in SEQ ID NOs.: 3, 4, 5, 6, 7, and 8. In another aspectof the invention features a binding molecule comprising the CDRs shownin SEQ ID NOs.: 3, 19, 5, 6, 7, and 8.

One aspect of the invention features a binding molecule comprising aheavy chain variable region comprising the amino acid sequence of SEQ IDNO.:1 and further comprising a light chain variable region comprisingthe amino acid sequence of SEQ ID NO.:2. Another aspect of the inventionfeatures a binding molecule comprising a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO.:66 and furthercomprising a light chain variable region comprising the amino acidsequence of SEQ ID NO.:2. In more than one embodiment, the bindingmolecule comprises human or substantially human heavy and light chainframework regions. In another embodiment, one or more human frameworkamino acid residues are mutated to the corresponding murine amino acidresidue. In another embodiment, the constant region comprises an IgG2bheavy chain constant region. In another embodiment, the constant regioncomprises a human, e.g., human IgG1, heavy chain constant region. Inanother embodiment, the binding molecule is altered to reduce effectorfunction and/or glycosylation. In one embodiment, the binding moleculebinds to human GITR In one embodiment, the binding molecule does notinduce apoptosis. In another embodiment, the binding molecule does notblock the primary mixed lymphocyte reaction. In yet another embodiment,the binding molecule abrogates the suppression of T effector cells by Tregulatory cells. In one embodiment, the binding molecule modulateseffector T cell proliferation. In one embodiment, the binding moleculeis murine. In another embodiment, the binding molecule comprises amurine IgG2b heavy chain. In one embodiment, the binding molecule is ahumanized antibody. In a further embodiment, the binding molecule is achimeric antibody. In yet another embodiment, the binding moleculemodulates the activity of human GITR. In another embodiment, the bindingmolecule attenuates degradation of I-κB in T cells.

Another aspect of the invention features a binding molecule that bindsto GITR on human T cells and human dendritic cells and has a bindingconstant (Kd) of 1×10⁻⁹ or less. In one embodiment, the binding moleculeabrogates the suppression of T effector cells by T regulatory cells. Inanother embodiment, the binding molecule is a humanized antibody.

Yet another aspect of the invention features a composition comprising abinding molecule of the invention and a pharmaceutically acceptablecarrier. In one embodiment, the composition further comprises at leastone additional therapeutic agent for treating cancer in a subject. Inone embodiment, the composition further comprises at least oneadditional therapeutic agent for treating a viral infection in asubject. In another embodiment, the composition further comprises atleast one tumor antigen for treating cancer in a subject. In yet anotherembodiment, the composition further comprises at least one antigen froma pathogenic agent.

One aspect of the invention features a method for abrogating thesuppression of T effector cells by T regulatory cells, comprisingcontacting human immune cells with a binding molecule of the inventionsuch that the suppression of T effector cells by T regulatory cells isabrogated.

Another aspect of the invention features a method for modulating T cellreceptor induced signaling in an effector T cell, comprising contactinga cell with a binding molecule of the invention, such that T cellinduced receptor signaling in an effector T cell is modulated. In oneembodiment, the method modulates the degradation of I-κB. In oneembodiment, the T cell is a Th1 cell. In another embodiment, the T cellis a CD4+ cell. In yet another embodiment, the T cell is a CD8+ cell.

Yet another aspect of the invention features a method for enhancing animmune response in a subject, comprising contacting a cell with abinding molecule of the invention such that that an immune response in asubject is enhanced.

Another aspect of the invention features a method for treating cancer ina subject, comprising contacting a cell with a binding molecule of theinvention such that cancer is treated in a subject. In one embodiment,the type of cancer is selected from the group consisting of: pancreaticcancer, melanomas, breast cancer, lung cancer, bronchial cancer,colorectal cancer, prostate cancer, stomach cancer, ovarian cancer,urinary bladder cancer, brain or central nervous system cancer,peripheral nervous system cancer, esophageal cancer, cervical cancer,uterine or endometrial cancer, cancer of the oral cavity or pharynx,liver cancer, kidney cancer, testicular cancer, biliary tract cancer,small bowel or appendix cancer, salivary gland cancer, thyroid glandcancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, and cancerof hematological tissues.

Another aspect of the invention features a method for treating aninfection caused by a pathogenic agent in a subject, comprisingcontacting a cell with the binding molecule of claim 1, such that theinfection caused by a pathogenic agent is treated in a subject. In oneembodiment, the pathogenic agent is a virus, e.g., selected from thegroup consisting of: hepatitis type A, hepatitis type B, hepatitis typeC, influenza, varicella, adenovirus, herpes simplex type I (HSV I),herpes simplex type II (HSV II), rinderpest, rhinovirus, echovirus,rotavirus, respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, hantavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV I), and human immunodeficiency virustype II (HIV II), any picornaviridae, enteroviruses, caliciviridae, anyof the Norwalk group of viruses, togaviruses, such as alphaviruses,flaviviruses, coronaviruses, rabies virus, Marburg viruses, ebolaviruses, parainfluenza virus, orthomyxoviruses, bunyaviruses,arenaviruses, reoviruses, rotaviruses, orbiviruses, human T cellleukemia virus type I, human T cell leukemia virus type II, simianimmunodeficiency virus, lentiviruses, polyomaviruses, parvoviruses,Epstein Barr virus, human herpesvirus 6, cercopithecine herpes virus 1(B virus), and poxviruses. In one embodiment, the method is used totreat a chronic viral infection.

In another embodiment, the pathogenic agent is a bacterium, e.g.,selected from the group consisting of: Neisseria spp, Streptococcus spp,S. mutans, Haemophilus spp., Moraxella spp, Bordetella spp,Mycobacterium spp, Legionella spp, Escherichia spp, Vibrio spp, Yersiniaspp, Campylobacter spp, Salmonella spp, Listeria spp., Helicobacter spp,Pseudomonas spp, Staphylococcus spp., Enterococcus spp, Clostridiumspp., Bacillus spp, Corynebacterium spp., Borrelia spp., Ehrlichia spp,Rickettsia spp, Chlamydia spp., Leptospira spp., Treponema spp.

Another aspect of the invention features a method for modulating GITRfunction comprising contacting human GITR with a binding molecule of theinvention in the presence of an immunostimulatory agent such that GITRfunction is modulated.

One aspect of the invention features a binding molecule comprising atleast one CDR amino acid sequence selected from the group consisting of:SEQ ID NO.:3, SEQ ID NO.:4, SEQ ID NO:19, SEQ ID NO.:5, SEQ ID NO.:6,SEQ ID NO.:7, and SEQ ID NO.:8. In one embodiment, the compositionfurther comprises at least one additional therapeutic agent for treatingcancer in a subject. In another embodiment, the binding moleculecomprises at least one CDR derived from the 6C8 binding molecule. Inanother embodiment, the binding molecule comprises at least two CDRsderived from the 6C8 binding molecule. In another embodiment, thebinding molecule comprises at least three CDRs derived from the 6C8binding molecule. In another embodiment, the binding molecule comprisesat least four CDRs derived from the 6C8 binding molecule. In anotherembodiment, the binding molecule comprises at least five CDRs derivedfrom the 6C8 binding molecule. In another embodiment, the bindingmolecule comprises at least six CDRs derived from the 6C8 bindingmolecule.

Another aspect of the invention features a binding molecule comprisingthe six CDRs shown in SEQ ID NOs.: 3, 4 or 19, 5, 6, 7, and 8.

Yet another aspect of the invention features a binding moleculecomprising a heavy chain variable region comprising the amino acidsequence of SEQ ID NO.:1 and further comprising a light chain variableregion comprising the amino acid sequence of SEQ ID NO.:2. In oneembodiment, a binding molecule comprises human or substantially humanheavy and light chain framework regions. In another embodiment a bindingmolecule of the invention comprises human framework regions in which oneor more human framework amino acid residues are backmutated to thecorresponding murine amino acid residue or are mutated to another aminoacid residue. In another embodiment, a binding molecule of the inventioncomprises a constant region of an immunoglobulin molecule, e.g., anIgG2b heavy chain constant region. In yet another embodiment, thebinding molecule binds to human GITR (hGITR). In one embodiment, thebinding molecule does not induce apoptosis. In another embodiment, thebinding molecule does not block the primary mixed lymphocyte reaction.In yet another embodiment, the binding molecule abrogates thesuppression of T effector cells by T regulatory cells. In oneembodiment, the binding molecule enhances effector T cell proliferation.In another embodiment, the binding molecule neutralizes the activity ofhuman GITR. In yet another embodiment, the binding molecule attenuatesdegradation of I-κB in T cells.

In one aspect, the invention features a binding molecule that binds toGITR on human T cells and human dendritic cells and has a bindingconstant (Kd) of 1×10⁻⁹ or less. In one embodiment, the binding moleculeabrogates the suppression of T regulatory cells. In another embodiment,the binding molecule is murine or comprises murine CDRs. In a furtherembodiment, the binding molecule comprises an IgG2b heavy chain. In oneembodiment, the binding molecule is a humanized antibody. In a furtherembodiment, the binding molecule is a chimeric antibody.

Another aspect of the invention features, a composition comprising abinding molecule of the invention and a pharmaceutically acceptablecarrier. In one embodiment, the composition further comprises at leastone additional therapeutic agent for treating cancer in a subject.

One aspect of the invention features a method for abrogating thesuppression of T effector cells by T regulatory cells, comprisingcontacting human immune cells with a binding molecule of the inventionsuch that the suppression of T regulatory cells is abrogated.

Another aspect of the invention features a method for modulating T cellreceptor induced signaling in an effector T cell, comprising contactinga cell with a binding molecule of the invention, such that T cellinduced receptor signaling in an effector T cell is modulated. In oneembodiment, the method modulates the degradation of I-κB. In oneembodiment, the T cell is a Th1 cell.

Yet another aspect of the invention features a method for enhancing animmune response in a subject, comprising contacting a cell with abinding molecule of the invention such that that an immune response in asubject is enhanced.

Another aspect of the invention features a method for treating cancer ina subject, comprising contacting a cell with a binding molecule of theinvention such that cancer is treated in a subject. In one embodiment,the type of cancer is selected from the group consisting of: pancreaticcancer, melanomas, breast cancer, lung cancer, bronchial cancer,colorectal cancer, prostate cancer, stomach cancer, ovarian cancer,urinary bladder cancer, brain or central nervous system cancer,peripheral nervous system cancer, esophageal cancer, cervical cancer,uterine or endometrial cancer, cancer of the oral cavity or pharynx,liver cancer, kidney cancer, testicular cancer, biliary tract cancer,small bowel or appendix cancer, salivary gland cancer, thyroid glandcancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, and cancerof hematological tissues.

Another aspect of the invention features a method for inhibiting GITRfunction comprising contacting human GITR with a binding molecule of theinvention in the presence of a stimulating agent such that GITR functionis inhibited.

One aspect of the invention features an isolated nucleic acid moleculecomprising a nucleotide sequence encoding a heavy chain variable regioncomprising the nucleotide sequence of SEQ ID NO.:9, optionallycomprising a leader sequence. Another aspect of the invention featuresan isolated nucleic acid molecule comprising a nucleotide sequenceencoding a heavy chain variable region comprising the nucleotidesequence of SEQ ID NO.:67, optionally comprising a leader sequence.

Another aspect of the invention features an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a light chainvariable region comprising the nucleotide sequence of SEQ ID NO.:10,optionally comprising a leader sequence.

Yet another aspect of the invention features an isolated nucleic acidmolecule comprising a nucleotide sequence encoding at least one CDRselected from the group consisting of: SEQ ID NO.:11, SEQ ID NO.:12 orSEQ ID NO:65, and SEQ ID NO.:13. In one embodiment, the isolated nucleicacid molecule comprises a nucleotide sequence encoding at least two CDRsderived from the 6C8 binding molecule. In another embodiment, theisolated nucleic acid molecule comprises a nucleotide sequence encodingat least three CDRs derived from the 6C8 binding molecule.

Another aspect of the invention features an isolated nucleic acidmolecule comprising a nucleotide sequence encoding at least one CDRselected from the group consisting of: SEQ ID NO.:14 SEQ ID NO.:15 andSEQ ID NO.:16. In one embodiment, the isolated nucleic acid moleculecomprises a nucleotide sequence encoding at least at least two CDRsderived from the 6C8 binding molecule. In another embodiment, theisolated nucleic acid molecule comprises a nucleotide sequence encodingat least three CDRs derived from the 6C8 binding molecule.

One aspect of the invention features an isolated nucleic acid moleculecomprising the nucleotide sequences shown in SEQ ID NOs.: 11-16 and SEQID NO:65.

One aspect of the invention features a recombinant expression vectorcomprising the nucleic acid molecules of the invention. In oneembodiment, a recombinant expression vector comprising a nucleic acidmolecule having a nucleotide sequence encoding the binding molecule ofthe invention is featured. In another embodiment, the invention featuresa host cell into which the recombinant expression vector of theinvention has been introduced. In another aspect the invention featuresa method for producing a binding molecule that binds human GITR,comprising culturing the host cell of the invention in a culture mediumuntil a binding molecule that binds human GITR is produced by the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an SDS-PAGE blot of purified mouse and human GITR bindingmolecules. Twelve micrograms of protein was loaded per well.

FIG. 2 depicts a size exclusion chromatography (SE-HPLC) of the purifiedhuman GITR binding molecule. Fifty micrograms of protein was injectedonto the SE-HPLC column at a flow rate of 0.6 ml/min. Purity of thebinding molecules by SE-HPLC yielded a population of binding moleculesin which 99.8% was in monomeric form and 0.2% aggregates.

FIG. 3 depicts a FACS analysis of L-M (mouse fibroblast) cellstransfected with the GITR gene that were stained with 50 μl ofsupernatant fluid from GITR-expressing hybridoma cells. The GITR bindingmolecule stained GITR-transfected cells but not the untransfected L-Mcells.

FIG. 4 depicts a FACS analysis demonstrating that GITR is primarilyexpressed on activated lymphocytes. The 6C8 binding molecules stainsCD4+, CD8+, CD25+ lymphocytes and very weakly stains CD103+ cells.

FIG. 5 depicts a saturation curve of the binding of the 6C8 bindingmolecule which was assessed by titrating biotin-labeled 6C8 onCD3-activated lymphocytes.

FIG. 6 is a graph showing that the 6C8 binding molecule is costimulatoryto T lymphocytes which are stimulated with sub-optimal OKT3 (anti-CD3;0.01 μg/ml) and incubated with either anti-CD28, or anti-GITR. Anisotype control (IgG2b) was also used.

FIGS. 7A and 7B are graphs demonstrating that the 6C8 binding moleculedoes not induce apoptosis. Lymphocytes were activated with PHA for 3days prior to the addition of 10 μg/ml of YTH655 (an anti-CD2 antibodyknown to induce apoptosis on activated lymphocytes; Friend, P., et al.(1987) Transplant. Proc. 19:4317), 6C8, or an isotype control (IgG2b).Apoptosis was measured by cell viability counts (A) and annexin Vstaining (B) and measuring apoptosis by flow cytometry.

FIG. 8 is a graph demonstrating that the 6C8 binding molecule does notblock a primary mixed lymphocyte reaction (MLR). Lymphocytes fromallogenic donors were mixed in the presence of TRX1 (anti-human CD4),6C8 or MOPC (an isotype control for TRX1) at various concentrations. Thecells were incubated for 3 days and pulsed with ³H-thymidine 18 hoursbefore the cells were harvested and counted.

FIG. 9 is a graph demonstrating that the 6C8 binding molecule blocks thesuppression of T effector cells induced by Treg cells. CD4+/CD25+ cellswere added to CD4+/CD25− cells at various ratios. The cells werestimulated with plate-bound anti-CD3 and anti-CD28. At ratios of 1:1there was inhibition of proliferation of the CD4+/CD25− cells. Theaddition of 6C8 at two different dilutions was able to block thesuppression of CD4+ T effector cells induced by the CD4+/CD25+ Tregulatory cells.

FIG. 10 is a graph demonstrating that the 6C8 binding molecule isco-stimulatory even when T cells are stimulated with anti-CD3 in theabsence of anti-CD28. CD4+/CD25+ cells were incubated with CD4+/CD25−cells at different cell ratios. The cells were stimulated withplate-bound anti-CD3 only. 6C8 was added to the cells and under theseconditions was co-stimulatory.

FIG. 11 is a graph demonstrating the effect of anti-GITR on I-κBdegradation in CD3 activated T cells.

FIG. 12 is a graph demonstrating the effect of anti-GITR on I-κBphosphorylation in CD3 activated T cells.

FIG. 13 is a graph demonstrating the effect of anti-GITR on I-κBdegradation, in CD3 plus CD28 activated T cells.

FIG. 14 is a graph demonstrating the effect of anti-GITR on I-κBphosphorylation, CD3 plus CD28 activated T cells.

FIG. 15 is a graph demonstrating that 6C8 and the R&D Systems(Minneapolis, Minn.) antibody recognize unique epitopes. The competitionassay was performed on both OKT3 and Con A activated lymphocytes. Oneμg/ml of 6C8 was used with various amounts of the competing R&D Systemsantibody (GITR/TNFRSF18 monoclonal antibody). There was some competitionobserved at the highest concentration of antibody, but this is mostlikely due to steric hindrance.

FIG. 16 shows the kinetic analysis of the 6C8 anti-GITR antibody versusthe R&D Systems GITR antibody.

FIG. 17 is a graph showing the percent survival of mice injected withmitomycin C treated B16 cells following treatment with anti-GITRantibody (2F8 rat anti-mouse GITR binding molecule).

FIGS. 18A-18D depict the nucleic acid and amino acid sequences of thevariable heavy chain (VHD) (A (SEQ ID NO: 9) and B (SEQ ID NO: 1),respectively) and variable light chain (VKA) (C (SEQ ID NO: 10) and D(SEQ ID NO: 2), respectively) of the 6C8 binding molecule. The leadersequences are shown in bold; the framework sequences are underlined; theCDR sequences are italicized.

FIGS. 19A and 19B are graphs showing that 2F8 and 2F8 F(ab′)2 fragmentsenhance the humoral response to HA.

FIGS. 20A and 20B are graphs showing that 2F8 and 2F8 F(ab′)2 fragmentsenhance the humoral response to Ova.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides binding molecules that specifically bindto GITR, e.g., human GITR (hGITR), on T cells and dendritic cells. Thebinding molecules of the invention are characterized by binding to hGITRwith high affinity, and in the presence of a stimulating agent, e.g.,CD3, they are agonistic, and they abrogate the suppression of T effector(Teff) cells by T regulatory (Treg) cells. Various aspects of theinvention relate to binding molecules, and pharmaceutical compositionsthereof, as well as nucleic acids encoding binding molecules,recombinant expression vectors and host cells for making such bindingmolecules. Methods of using a binding molecule of the invention todetect human GITR or to modulate human GITR activity, either in vitro orin vivo, are also encompassed by the invention.

In order that the present invention may be more readily understood,certain terms are first defined.

I. Definitions

The term “glucocorticoid-induced TNF receptor” (abbreviated herein as“GITR”), also known as TNF receptor superfamily 18 (TNFRSF18), as usedherein, refers to a member of the tumor necrosis factor/nerve growthfactor receptor family. It is a 241 amino acid type I transmembraneprotein characterized by three cysteine pseudorepeats in theextracellular domain and specifically protects T-cell receptor-inducedapoptosis, although it does not protect cells from other apoptoticsignals, including Fas triggering, dexamethasone treatment, or UVirradiation (Nocentini, G, et al. (1997) Proc. Natl. Acad. Sci., USA94:6216-622). The nucleic acid sequence of human GITR (hGITR) is setforth in SEQ ID NO.: 17 and the amino acid sequence is set forth in SEQID NO.: 18.

The term “binding molecule” as used herein includes molecules thatcontain at least one antigen binding site that specifically binds toGITR. By “specifically binds” it is meant that the binding moleculesexhibit essentially background binding to non-GITR molecules. Anisolated binding molecule that specifically binds GITR may, however,have cross-reactivity to GITR molecules from other species.

The binding molecules of the invention may comprise an immunoglobulinheavy chain of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. Binding molecules may have both a heavy and alight chain. As used herein, the term binding molecule also includes,antibodies (including full length antibodies), monoclonal antibodies(including full length monoclonal antibodies), polyclonal antibodies,multi specific antibodies (e.g., bispecific antibodies), human,humanized or chimeric antibodies, and antibody fragments, e.g., Fabfragments, F(ab′) fragments, fragments produced by a Fab expressionlibrary, epitope-binding fragments of any of the above, and engineeredforms of antibodies, e.g., scFv molecules, so long as they exhibit thedesired activity, e.g., binding to GITR.

An “antigen” is an entity (e.g., a proteinaceous entity or peptide) towhich a binding molecule specifically binds.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which a binding molecule specifically binds. Epitopes can beformed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in aunique spatial conformation. Methods of determining spatial conformationof epitopes include, for example, X-ray crystallography and2-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed.(1996).

Binding molecules that recognize the same epitope can be identified in asimple immunoassay showing the ability of one antibody to block thebinding of another antibody to a target antigen, i.e., a competitivebinding assay. Competitive binding is determined in an assay in whichthe binding molecule being tested inhibits specific binding of areference binding molecule to a common antigen, such as GITR. Numeroustypes of competitive binding assays are known, for example: solid phasedirect or indirect radioimmunoassay (MA); solid phase direct or indirectenzyme immunoassay (EIA) sandwich competition assay (see Stahli et al.,Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidinEIA (see Kirkland et al., J. Immunol. 137:3614 (1986)); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborPress (1988)); solid phase direct label RIA using I-125 label (see Morelet al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidinEIA (Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically, suchan assay involves the use of purified antigen bound to a solid surfaceor cells bearing either of these, an unlabeled test binding molecule anda labeled reference binding molecule. Competitive inhibition is measuredby determining the amount of label bound to the solid surface or cellsin the presence of the test binding molecule. Usually the test bindingmolecule is present in excess. Usually, when a competing bindingmolecule is present in excess, it will inhibit specific binding of areference binding molecule to a common antigen by at least 50-55%,55-60%, 60-65%, 65-70% 70-75% or more.

An epitope is also recognized by immunologic cells, for example, B cellsand/or T cells. Cellular recognition of an epitope can be determined byin vitro assays that measure antigen-dependent proliferation, asdetermined by ³H-thymidine incorporation, by cytokine secretion, byantibody secretion, or by antigen-dependent killing (cytotoxic Tlymphocyte assay). The term “monoclonal binding molecule” as used hereinrefers to a binding molecule obtained from a population of substantiallyhomogeneous binding molecules. Monoclonal binding molecules are highlyspecific, being directed against a single antigenic site. Furthermore,in contrast to polyclonal binding molecule preparations which typicallyinclude different binding molecules directed against differentdeterminants (epitopes), each monoclonal binding molecule is directedagainst a single determinant on the antigen. The modifier “monoclonal”indicates the character of the binding molecule as being obtained from asubstantially homogeneous population of binding molecules, and is not tobe construed as requiring production of the binding molecule by anyparticular method. For example, the monoclonal binding molecules to beused in accordance with the present invention may be made by thehybridoma method first described by Kohler, et al., Nature 256:495(1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat.No. 4,816,567). The “monoclonal binding molecules” may also be isolatedfrom phage antibody libraries using the techniques described inClackson, et al., Nature 352:624-628 (1991) and Marks et al., J. MolBiol. 222:581-597 (1991), for example.

The term “chimeric binding molecule” refers to a binding moleculecomprising amino acid sequences derived from different species. Chimericbinding molecules can be constructed, for example by geneticengineering, from binding molecule gene segments belonging to differentspecies.

The monoclonal binding molecules herein specifically include “chimeric”binding molecules in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in bindingmolecules derived from a particular species or belonging to a particularantibody class or subclass, while the remainder of the chain(s) isidentical with or homologous to corresponding sequences in bindingmolecules derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such binding molecules, solong as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; and Morrison, et al., Proc. Natl. Acad. Sci. USA 81:6851-6855(1984)). e.g., binding to human GITR (hGITR).

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CL) and the heavy chain (CH1, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminus is a variable region and at theC-terminus is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

A “variable region” when used in reference to a binding molecule refersto the amino terminal portion of a binding molecule which confersantigen binding onto the molecule and which is not the constant region.The term includes functional fragments thereof which maintain some orall of the binding function of the whole variable region.

The term “hypervariable region” when used herein refers to the regionsof a binding molecule variable domain which are hypervariable insequence and/or form structurally defined loops. The hypervariableregion comprises amino acid residues from a “complementarity determiningregion” or “CDR”.

As used herein, the term “CDR” or “complementarity determining region”means the noncontiguous antigen combining sites found within thevariable region of both heavy and light chain polypeptides. Theseparticular regions have been described by Kabat, et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat, et al., Sequences of protein ofimmunological interest. (1991), and by Chothia, et al., J. Mol. Biol.196:901-917 (1987) and by MacCallum, et al., J. Mol. Biol. 262:732-745(1996) where the definitions include overlapping or subsets of aminoacid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of a binding moleculeor grafted binding molecule or variants thereof is within the scope ofthe term as defined and used herein.

As used herein, the term “framework region” or “FR” means each domain ofthe framework that is separated by the CDRs. Therefore, a variableregion framework is between about 100-120 amino acids in length butrefers only those amino acids outside of the CDRs.

“Humanized” forms of non-human (e.g., murine) binding molecules arechimeric antibodies which contain minimal sequence derived fromnon-human binding molecule. For the most part, humanized bindingmolecules are human binding molecules (acceptor/recipient bindingmolecule) in which residues from a hyper-variable region are replaced byresidues from a hypervariable region of a non-human species (donorbinding molecule) such as mouse, rat, rabbit or nonhuman primate havingthe desired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human binding molecule arealtered, e.g., replaced by, substituted, or backmutated to correspondingnon-human residues. Furthermore, humanized binding molecules maycomprise residues which are not found in the recipient binding moleculeor in the donor binding molecule. These modifications are generally madeto further refine binding molecule performance. In general, thehumanized binding molecule will comprise substantially all of at leastone, and typically two, variable domains, in which all or substantiallyall of the hypervariable loops correspond to those of a non-humanbinding molecule and all or substantially all of the FR regions arethose of a human binding molecule sequence. The humanized bindingmolecule optionally also will comprise at least a portion of a bindingmolecule constant region (Fc), typically that of a human bindingmolecule. For further details, see Jones, et al., Nature 321:522-525(1986); Riechmann, et al., Nature 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol. 2:593-596 (1992).

Preferably, a humanized binding molecule of the invention comprises atleast one CDR selected from the group consisting of SEQ ID NO.:3(GFSLSTSGMGVG (HC CDR1)), SEQ ID NO.:4 (HIWWDDDKYYNPSLKS (HC CDR2N)),SEQ ID NO.:5 (TRRYFPFAY (HC CDR3)), SEQ ID NO.:6 (KASQNVGTNVA (LCCDR1)), SEQ ID NO.:7 (SASYRYS (LC CDR2)), SEQ ID NO.:8 (QQYNTDPLT (LCCDR3)), and SEQ ID NO:19 (HIWWDDDKYYQPSLKS (HC CDR2Q)).

The term “engineered” or “recombinant” binding molecule, as used hereinincludes binding molecules that are prepared, expressed, created orisolated by recombinant means, such as binding molecules expressed usinga recombinant expression vector transfected into a host cell, bindingmolecules isolated from a recombinant, combinatorial binding moleculelibrary, binding molecules isolated from an animal (e.g., a mouse) thatis transgenic for human immunoglobulin genes (see e.g., Taylor, L. D.,et al. (1992) Nucl. Acids Res. 20:6287-6295) or binding moleculesprepared, expressed, created or isolated by any other means thatinvolves splicing of human binding molecule gene sequences to other DNAsequences. In certain embodiments, however, such recombinant humanbinding molecules are subjected to in vitro mutagenesis (or, when ananimal transgenic for human Ig sequences is used, in vivo somaticmutagenesis) and thus the amino acid sequences of the VH and VL regionsof the recombinant binding molecules are sequences that, while derivedfrom and related to human germline VH and VL sequences, may notnaturally exist within the human binding molecule germline repertoire invivo.

An “isolated binding molecule”, as used herein, refers to a bindingmolecule that is substantially free of other binding molecules havingdifferent antigenic specificities (e.g., an isolated binding moleculethat specifically binds GITR is substantially free of binding moleculesthat specifically bind antigens other than GITR). Moreover, an isolatedbinding molecule may be substantially free of other cellular materialand/or chemicals. An “isolated” binding molecule is one which has beenidentified and separated and/or recovered from a component of itsnatural environment. Contaminant components of its natural environmentinclude, e.g., materials which would interfere with diagnostic ortherapeutic uses for the binding molecule, and may include enzymes,hormones, and other proteinaceous or nonproteinaceous solutes. Inpreferred embodiments, the binding molecule will be purified (1) togreater than 95% by weight of binding molecule as determined by theLowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated bindingmolecules include binding molecules in situ within recombinant cellssince at least one component of the binding molecule's naturalenvironment will not be present. Ordinarily, however, isolated bindingmolecules will be prepared by at least one purification step.

As used herein the term “binding constant” “(kd)”, also referred to as“affinity constant”, is a measure of the extent of a reversibleassociation between two molecular species includes both the actualbinding affinity as well as the apparent binding affinity. The actualbinding affinity is determined by calculating the ratio of the Kassoc inM⁻¹S⁻¹ to the Kdissoc in S⁻¹ and has the units “M⁻¹”. Therefore,conferring or optimizing binding affinity includes altering either orboth of these components to achieve the desired level of bindingaffinity. The apparent affinity can include, for example, the avidity ofthe interaction. For example, a bivalent heteromeric variable regionbinding fragment can exhibit altered or optimized binding affinity dueto its valency. Binding affinity can be determined by measurement ofsurface plasmon resonance, e.g., using a BIAcore system.

The term “nucleic acid molecule”, as used herein, includes DNA moleculesand RNA molecules. A nucleic acid molecule may be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

The term “isolated nucleic acid molecule”, as used herein in referenceto nucleic acids encoding binding molecules that bind GITR, refers to anucleic acid molecule in which the nucleotide sequences encoding thebinding molecule are free of other nucleotide sequences which othersequences may naturally flank the nucleic acid in human genomic DNA.These sequences may optionally include 5′ or 3′nucleotide sequencesimportant for regulation or protein stability.

The term “vector”, as used herein, refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention includes such other forms ofexpression vectors, such as viral vectors (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), which serveequivalent functions.

The term “recombinant host cell” (or simply “host cell”), as usedherein, refers to a cell into which a recombinant expression vector hasbeen introduced. It should be understood that such terms are intended torefer not only to the particular subject cell but to the progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term “host cell” as used herein.

As used herein, the term “T cell” (i.e., T lymphocyte) includes allcells within the T cell lineage, including thymocytes, immature T cells,mature T cells and the like, from a mammal (e.g., human). Preferably, Tcells are mature T cells that express either CD4 or CD8, but not both,and a T cell receptor. The various T cell populations described hereincan be defined based on their cytokine profiles and their function, andare known to one of skill in the art.

As used herein, the term “dendritic cell” refers to professionalantigen-presenting cells (APCs) capable of activating naïve T cells andstimulating the growth and differentiation of B cells.

As used herein, the term “naïve T cells” includes T cells that have notbeen exposed to cognate antigen and so are not activated or memorycells. Naïve T cells are not cycling and human naïve T cells areCD45RA+. If naïve T cells recognize antigen and receive additionalsignals depending upon but not limited to the amount of antigen, routeof administration and timing of administration, they may proliferate anddifferentiate into various subsets of T cells, e.g. effector T cells.

As used herein, the term “effector T cell” or “Teff cell” includes Tcells which function to eliminate antigen (e.g., by producing cytokineswhich modulate the activation of other cells or by cytotoxic activity).The term “effector T cell” includes T helper cells (e.g., Th1 and Th2cells) and cytotoxic T cells. Th1 cells mediate delayed typehypersensitivity responses and macrophage activation while Th2 cellsprovide help to B cells and are critical in the allergic response(Mosmann and Coffman, 1989, Annu. Rev. Immunol. 7, 145-173; Paul andSeder, 1994, Cell 76, 241-251; Arthur and Mason, 1986, J. Exp. Med. 163,774-786; Paliard, et al., 1988, J. Immunol. 141, 849-855; Finkelman, etal., 1988, J. Immunol. 141, 2335-2341).

As used herein, the term “T helper type 1 response” (Th1 response)refers to a response that is characterized by the production of one ormore cytokines selected from IFN-γ, IL-2, TNF, and lymphotoxin (LT) andother cytokines produced preferentially or exclusively by Th1 cellsrather than by Th2 cells. As used herein, a “T helper type 2 response”(Th2 response) refers to a response by CD4+ T cells that ischaracterized by the production of one or more cytokines selected fromIL-4, IL-5, IL-6 and IL-10, and that is associated with efficient B cell“help” provided by the Th2 cells (e.g., enhanced IgG1 and/or IgEproduction).

As used herein, the term “regulatory T cell” or “Treg cell” includes Tcells which produce low levels of IL-2, IL-4, IL-5, and IL-12.Regulatory T cells produce TNFα, TGFβ, IFN-γ, and IL-10, albeit at lowerlevels than effector T cells. Although TGFβ is the predominant cytokineproduced by regulatory T cells, the cytokine is produced at levels lessthan or equal to that produced by Th1 or Th2 cells, e.g., an order ofmagnitude less than in Th1 or Th2 cells. Regulatory T cells can be foundin the CD4+CD25+ population of cells (see, e.g., Waldmann and Cobbold.2001. Immunity. 14:399). Regulatory T cells actively suppress theproliferation and cytokine production of Th1, Th2, or näive T cellswhich have been stimulated in culture with an activating signal (e.g.,antigen and antigen presenting cells or with a signal that mimicsantigen in the context of MHC, e.g., anti-CD3 antibody, plus anti-CD28antibody).

As used herein, the term “tolerance” includes refractivity to activatingreceptor-mediated stimulation. Such refractivity is generallyantigen-specific and persists after exposure to the tolerizing antigenhas ceased. For example, tolerance is characterized by lack of cytokineproduction, e.g., IL-2, or can be assessed by use of a mixed lymphocyteculture assay. Tolerance can occur to self antigens or to foreignantigens.

A “mixed lymphocyte culture” (“MLC”) is a type of lymphocyteproliferation test in which lymphocytes from two individuals arecultured together and the proliferative response (“mixed lymphocytereaction”) is measured by ³H-labeled thymidine uptake.

As used herein, the term “apoptosis” also referred to as programmed celldeath (PCD), is the death of a cell characterized by features including,but not limited to, condensation of nuclear heterochromatin, cellshrinkage, cytoplasmic condensation, and in a later stage of apoptosis,endonuclease mediated cleavage of the DNA of the cell into discretefragments. Upon electrophoretic analysis of the DNA of a cell in whichapoptosis has occurred, a characteristic “ladder” of discrete DNAfragments may be apparent.

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment may include thosealready having a disorder as well as those which do not yet have adisorder.

A “disorder” is any condition that would benefit from treatment with abinding molecule of the present invention. This includes chronic andacute disorders or diseases or pathological conditions associated withimmune responses that are too high or too low.

Various aspects of the invention are described in further detail in thefollowing subsections.

II. GITR Binding Molecules

The present invention provides isolated GITR binding molecules.Exemplary binding molecules of the present invention include the 6C8antibody and the 2F8 antibody. The 6C8 antibody is an anti-GITR antibodythat binds to GITR on T cells and dendritic cells, e.g., human T cellsand dendritic cells, with high affinity. Preferably, such bindingmolecules abrogate the suppression of Teff cells by Treg cells and areagonistic to partially activated T cells in vitro in the presence of astimulating agent, e.g., CD3.

In one embodiment, the a VH domain of a binding molecule of theinvention comprises the amino acid sequence set forth in SEQ ID NO:1.(6C8 VH domain “N”, including leader). It will be understood thatalthough some of the sequences of binding molecules described hereininclude leader sequences, a binding molecule of the invention may alsoexclude the leader sequence, which is optional. For example, in oneembodiment, a binding molecule of the invention comprises the amino acidsequence of the mature protein shown in SEQ ID NO:1. e.g., amino acids20-138 of SEQ ID NO:1.

In one embodiment, the a VH domain of a binding molecule of theinvention comprises the amino acid sequence set forth in SEQ ID NO:66.(6C8 VH domain “Q”, including leader).

In one embodiment, the a VL domain of a binding molecule of theinvention comprises the amino acid sequence set forth in SEQ ID NO:2.(6C8 VL domain, including leader).

In one embodiment, the a VH domain of a binding molecule of theinvention comprises amino acid residues 20-138 of SEQ ID NO.:1. (6C8 VHdomain “N”, without leader).

In one embodiment, the a VH domain of a binding molecule of theinvention comprises amino acid residues 20-138 of SEQ ID NO.:66. (6C8 VHdomain “Q”, without leader).

In one embodiment, the a VL domain of a binding molecule of theinvention comprises comprises amino acid residues 21-127 of SEQ IDNO.:2. (6C8 VL domain, without leader).

In one embodiment of the invention the VL chain comprises a leaderand/or signal sequence, i.e., amino acid residues 1-20 of SEQ ID NO:2(SEQ ID NO:59). In one embodiment, the VH chain comprises a leaderand/or signal sequence, i.e., amino acid residues 1-19 of SEQ ID NO:1(SEQ ID NO:64).

In one embodiment, a binding molecule of the invention comprisies a VHdomain comprising a CDR set forth in SEQ ID NO:3. (6C8 VH CDR1).

In one embodiment, a binding molecule of the invention comprisies a VHdomain comprising a CDR set forth in SEQ ID NO:4. (6C8 VH CDR2-“N”).

In one embodiment, a binding molecule of the invention comprisies a VHdomain comprising a CDR set forth in SEQ ID NO:5. (6C8 VH CDR3).

In one embodiment, a binding molecule of the invention comprisies a VHdomain comprising a CDR set forth in SEQ ID NO:19. (6C8 VHCDR2-alternate “Q”).

In one embodiment, a binding molecule of the invention comprisies a VLdomain comprising a CDR set forth in SEQ ID NO:6. (6C8 VL CDR1).

In one embodiment, a binding molecule of the invention comprisies a VLdomain comprising a CDR set forth in SEQ ID NO:7. (6C8 VL CDR2).

In one embodiment, a binding molecule of the invention comprisies a VLdomain comprising a CDR set forth in SEQ ID NO:8. (6C8 VL CDR3).

The invention also pertains to nucleic acid molecules encoding the aboveamino acid sequences.

In one embodiment, the a VH domain of a binding molecule of theinvention comprises the nucleotide sequence set forth in SEQ ID NO:9.(6C8 VH domain, “N”, including leader).

In one embodiment, the a VH domain of a binding molecule of theinvention comprises the nucleotide sequence set forth in SEQ ID NO:65.(6C8 VH domain, “Q”, including leader).

In one embodiment, the a VH domain of a binding molecule of theinvention comprises nucleotides 58-414 of SEQ ID NO.:9. (6C8 VH domain,“N”, without leader).

In one embodiment, the a VH domain of a binding molecule of theinvention comprises nucleotides 58-414 of SEQ ID NO.:65. (6C8 VH domain,“Q”, without leader).

In one embodiment, the a VL domain of a binding molecule of theinvention comprises the nucleotide sequence set forth in SEQ ID NO:10.(6C8 VL domain, including leader).

In one embodiment, the a VL domain of a binding molecule of theinvention comprises nucleotides 61-381 of SEQ ID NO.:10. (6C8 VL domain,without leader).

In one embodiment, a binding molecule of the invention comprises a VHdomain comprising a CDR the nucleic acid sequence of which is set forthin SEQ ID NO:11. (6C8 VH CDR1).

In one embodiment, a binding molecule of the invention comprises a VHdomain comprising a CDR the nucleic acid sequence of which is set forthin SEQ ID NO:12. (6C8 VH CDR2-“AAT”).

In one embodiment, a binding molecule of the invention comprises a VHdomain comprising a CDR the nucleic acid sequence of which is set forthin SEQ ID NO:13. (6C8 VH CDR3).

In one embodiment, a binding molecule of the invention comprises a VHdomain comprising a CDR the nucleic acid sequence of which is set forthin SEQ ID NO:65. (6C8 VH CDR2-alternate “CAA”).

In one embodiment, a binding molecule of the invention comprises a VLdomain comprising a CDR the nucleic acid sequence of which is set forthin SEQ ID NO:14. (6C8 VL CDR1).

In one embodiment, a binding molecule of the invention comprises a VLdomain comprising a CDR the nucleic acid sequence of which is set forthin SEQ ID NO:15. (6C8 VL CDR2).

In one embodiment, a binding molecule of the invention comprises a VLdomain comprising a CDR the nucleic acid sequence of which is set forthin SEQ ID NO:16. (6C8 VL CDR3).

In one embodiment, the a CL domain of a binding molecule of theinvention comprises the amino acid sequence set forth in SEQ ID NO:20.(Murine IgG2a light chain constant region).

In one embodiment, the a CH domain of a binding molecule of theinvention comprises the amino acid sequence set forth in SEQ ID NO:21.(Murine IgG2a heavy chain constant region).

In one embodiment, a binding molecule of the invention comprises theamino acid sequence set forth in SEQ ID NO:22. (Chimeric-6C8 VL/human CLIgG1).

In one embodiment, a binding molecule of the invention comprises theamino acid sequence set forth in SEQ ID NO:23. (Chimeric Gly-6C8VH/human CH IgG1).

In one embodiment, a binding molecule of the invention comprises theamino acid sequence set forth in SEQ ID NO:24. (Chimeric Agly-6C8VH/human CH IgG1).

In one embodiment, a binding molecule of the invention comprises theamino acid sequence set forth in SEQ ID NO:44. (Humanized 6C8 VL).

In one embodiment, a binding molecule of the invention comprises theamino acid sequence set forth in SEQ ID NO:53. (Humanized 6C8 VH “N”).

In one embodiment, a binding molecule of the invention comprises theamino acid sequence set forth in SEQ ID NO:54. (Humanized 6C8 VH “Q”).

In one embodiment, the a CL domain of a binding molecule of theinvention comprises the amino acid sequence set forth in SEQ ID NO:55.(Human IgG1 Gly heavy chain constant region).

In one embodiment, the a CH domain of a binding molecule of theinvention comprises the amino acid sequence set forth in SEQ ID NO:56.(Human IgG1 Agly heavy chain constant region).

In one embodiment, the a CL domain of a binding molecule of theinvention comprises the amino acid sequence set forth in SEQ ID NO:57.(Human IgG1 light chain constant region).

In one embodiment, a binding molecule of the invention comprises theamino acid sequence set forth in SEQ ID NO:58. (Complete Humanized 6C8Light).

In one embodiment, a binding molecule of the invention comprises theamino acid sequence set forth in SEQ ID NO:60. (Complete Humanized 6C8Heavy-HuN6C8-Gly).

In one embodiment, a binding molecule of the invention comprises theamino acid sequence set forth in SEQ ID NO:61. (Complete Humanized 6C8Heavy-HuN6C8-Agly).

In one embodiment, a binding molecule of the invention comprises theamino acid sequence set forth in SEQ ID NO:62. (Complete Humanized 6C8Heavy-HuQ6C8-Gly).

In one embodiment, a binding molecule of the invention comprises theamino acid sequence set forth in SEQ ID NO:63. (Complete Humanized 6C8Heavy-HuQ6C8-Agly).

In one embodiment, a binding molecule of the invention has VL and VHsequences as shown in FIGS. 18A-18D; the amino acid sequence of the 6C8VH region is also shown in SEQ ID NO: 1; the amino acid sequence of the6C8 VL region is shown in SEQ ID NO: 2. In another embodiment, a bindingmolecule of the invention has LC and HC sequences as set forth in SEQ IDNOs:20 and 21;

(SEQ ID NO: 20) ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIV KSFNRNE; (SEQ ID NO:21) AKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALLQSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECKCPAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAQVYILPPPAEQLSRKDVSLTCLVVGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTSKWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK.

In one embodiment of the invention the VL chain comprises a leaderand/or signal sequence, e.g., amino acid residues 1-20 of SEQ ID NO:2.In one embodiment, the VH chain comprises a leader and/or signalsequence, e.g., amino acid residues 1-19 of SEQ ID NO:1. In anotherembodiment, a binding molecule of the invention does not comprise aleader and/or signal sequence.

In one aspect, the invention pertains to 6C8 binding molecules and otherbinding molecules with equivalent properties to 6C8, such as highaffinity binding to GITR and abrogation of suppression of Teff cells byTreg cells. In addition, the binding molecules of the invention do notinduce apoptosis, nor do they inhibit a mixed lymphocyte reaction.Accordingly, equivalent binding molecules of the invention are GITRagonists, i.e., they induce signaling via GITR. GITR is a member of theTNFR superfamily. Since members of the TNFR family are involved in cellsurvival and apoptosis by signaling through NF-κB, in one embodiment,the binding molecules of the present invention attenuate degradation ofI-κB.

In one embodiment, the invention provides isolated hGITR bindingmolecules with a light chain variable region (VL) comprising the aminoacid sequence of SEQ ID NO: 2, and optionally a leader sequence, and aheavy chain variable region (VH) comprising the amino acid sequence ofSEQ ID NO: 1, and optionally a leader sequence. In certain embodiments,a binding molecule comprises a heavy chain constant region, such as anIgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant region.Furthermore, the binding molecule can comprise a light chain constantregion, either a kappa light chain constant region or a lambda lightchain constant region. Preferably, the binding molecule comprises akappa light chain constant region. In one embodiment, a binding moleculeof the invention comprises a light chain constant region as set forth inSEQ ID NO:20. In one embodiment, a binding molecule of the inventioncomprises a heavy chain constant region as set forth in SEQ ID NO:21. Inone embodiment, a binding molecule of the invention comprises a heavychain constant region as set forth in SEQ ID NO:55. In one embodiment, abinding molecule of the invention comprises a heavy chain constantregion as set forth in SEQ ID NO:56. In one embodiment, a bindingmolecule of the invention comprises a heavy chain constant region as setforth in SEQ ID NO:57.

In another embodiment, the invention provides a binding molecule having6C8-related VL CDR domains, for example, binding molecules with a lightchain variable region (VL) having at least one CDR domain comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8. In another embodiment, a light chainvariable region (VL) has at least two CDR domains comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 6, SEQ IDNO: 7, SEQ ID NO: 8. In yet another embodiment, a light chain variableregion (VL) has CDR domains comprising the amino acid sequencesconsisting of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8.

In still other embodiments, the invention provides a binding moleculehaving 6C8-related VH CDR domains, for example, binding molecules with alight chain variable region (VH) having a CDR domain comprising an aminoacid sequence selected from the group consisting of SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, and SEQ ID NO:19. In another embodiment, a heavychain variable region (VH) has at least two CDR domains comprising anamino acid sequence selected from the group consisting of SEQ ID NO: 3,SEQ ID NO: 4, SEQ ID NO: 5, and SEQ ID NO:19. In yet another embodiment,a heavy chain variable region (VH) has CDR domains comprising the aminoacid sequences consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5,and SEQ ID NO:19.

In another embodiment, a binding molecule of the invention comprises atleast one CDR derived from a murine anti-human GITR binding molecule,e.g., a 6C8 binding molecule. As used herein the term “derived from” adesignated protein refers to the origin of the polypeptide. In oneembodiment, the polypeptide or amino acid sequence which is derived froma particular starting polypeptide is a CDR sequence or sequence relatedthereto. In another embodiment, the polypeptide or amino acid sequencewhich is derived from a particular starting polypeptide is a FR sequenceor sequence related thereto. In one embodiment, the amino acid sequencewhich is derived from a particular starting polypeptide is notcontiguous.

For example, in one embodiment, one, two, three, four, five, or six CDRsare derived from a murine 6C8 antibody. In one embodiment, a bindingmolecule of the invention comprises at least one heavy or light chainCDR of a murine 6C8 antibody. In another embodiment, a binding moleculeof the invention comprises at least two CDRs from a murine 6C8 antibody.In another embodiment, a binding molecule of the invention comprises atleast three CDRs from a murine 6C8 antibody. In another embodiment, abinding molecule of the invention comprises at least four CDRs from amurine 6C8 antibody. In another embodiment, a binding molecule of theinvention comprises at least five CDRs from a murine 6C8 antibody. Inanother embodiment, a binding molecule of the invention comprises atleast six CDRs from a murine 6C8 antibody.

It will also be understood by one of ordinary skill in the art that abinding molecule of the invention may be modified such that they vary inamino acid sequence from the 6C8 molecule from which they were derived.For example, nucleotide or amino acid substitutions leading toconservative substitutions or changes at “non-essential” amino acidresidues may be made (e.g., in CDR and/or framework residues) andmaintain the ability to bind to GITR, e.g., human GITR.

In one embodiment, the at least one CDR (or at least one CDR from thegreater than one 6C8 CDRs that are present in the binding molecule) ismodified to vary in sequence from the CDR of a naturally occurring 6C8binding molecule, yet retains the ability to bind to 6C8. For example,in one embodiment, one or more CDRs from a 6C8 antibody are modified toremove potential glycosylation sites. For example, since the amino acidsequence Asn-X-(Ser/Thr) is a putative consensus sequence for aglycosylation site which may affect the production of the bindingmolecule, and CDR2 of the 6C8 heavy chain has the sequence Asn-Pro-Ser,a second version of the heavy chain was prepared to conservativelysubstitute a glutamine (Gln) for an asparagine (Asn) at amino acidresidue 62 of SEQ ID NO:53.

In one embodiment, a binding molecule of the invention comprises apolypeptide or amino acid sequence that is essentially identical to thatof a 6C8 antibody, or a portion thereof wherein the portion consists ofat least 3-5 amino acids, of at least 5-10 amino acids, at least 10-20amino acids, at least 20-30 amino acids, or at least 30-50 amino acids,or which is otherwise identifiable to one of ordinary skill in the artas having its origin in the starting sequence.

In another embodiment, the polypeptide or amino acid sequence which isderived from a particular starting polypeptide or amino acid sequenceshares an amino acid sequence identity that is about 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or which isotherwise identifiable to one of ordinary skill in the art as having itsorigin in the starting sequence.

An isolated nucleic acid molecule encoding a non-natural variant of apolypeptide can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofthe binding molecule such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.Mutations may be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. In oneembodiment, conservative amino acid substitutions are made at one ormore non-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art,including basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a nonessential amino acid residue in a bindingmolecule polypeptide may be replaced with another amino acid residuefrom the same side chain family. In another embodiment, a string ofamino acids can be replaced with a structurally similar string thatdiffers in order and/or composition of side chain family members.

Alternatively, in another embodiment, mutations may be introducedrandomly along all or part of the binding molecule coding sequence.

Preferred binding molecules of the invention comprise framework andconstant region amino acid sequences derived from a human amino acidsequence. However, binding molecules may comprise framework and/orconstant region sequences derived from another mammalian species. Forexample, a primate framework region (e.g., non-human primate), heavychain portion, and/or hinge portion may be included in the subjectbinding molecules. In one embodiment, one or more murine amino acids maybe present in the framework region of a binding polypeptide, e.g., ahuman or non-human primate framework amino acid sequence may compriseone or more amino acid substitutions and/or backmutations in which thecorresponding murine amino acid residue is present. Preferred bindingmolecules of the invention are less immunogenic than the starting 6C8murine antibody.

The present invention also features chimeric and/or humanized bindingmolecules (i.e., chimeric and/or humanized immunoglobulins) specific forGITR. Chimeric and/or humanized binding molecules have the same orsimilar binding specificity and affinity as a mouse or other nonhumanbinding molecules that provide the starting material for construction ofa chimeric or humanized binding molecule.

A chimeric binding molecule is one whose light and heavy chain geneshave been constructed, typically by genetic engineering, fromimmunoglobulin gene segments belonging to different species. Forexample, the variable (V) segments of the genes from a mouse monoclonalbinding molecule may be joined to human constant (C) segments, such asIgG1 or IgG4. Human isotype IgG1 is preferred. An exemplary chimericbinding molecule is thus a hybrid protein consisting of the V orantigen-binding domain from a mouse binding molecule and the C oreffector domain from a human binding molecule.

In one embodiment, the invention pertains to humanized variable regionsof the 6C8 binding molecule and polypeptides comprising such humanizedvariable regions. In one embodiment, a binding molecule of the inventioncomprises at least one humanized 6C8 binding molecule variable region,e.g., a light chain or heavy chain variable region.

The term “humanized binding molecule” refers to a binding moleculecomprising at least one chain comprising variable region frameworkresidues derived from a human binding molecule chain (referred to as theacceptor immunoglobulin or binding molecule) and at least onecomplementarity determining region derived from a mouse-bindingmolecule, (referred to as the donor immunoglobulin or binding molecule).Humanized binding molecules can be produced using recombinant DNAtechnology, which is discussed below. See for example, e.g., Hwang, W.Y. K., et al. (2005) Methods 36:35; Queen et al., Proc. Natl. Acad. Sci.USA, (1989), 86:10029-10033; Jones et al., Nature, (1986), 321:522-25;Riechmann et al., Nature, (1988), 332:323-27; Verhoeyen et al., Science,(1988), 239:1534-36; Orlandi et al., Proc. Natl. Acad. Sci. USA, (1989),86:3833-37; U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,761;5,693,762; 6,180,370, Selick et al., WO 90/07861, and Winter, U.S. Pat.No. 5,225,539 (incorporated by reference in their entirety for allpurposes). The constant region(s), if present, are preferably is alsoderived from a human immunoglobulin.

When a preferred non-human donor binding molecule has been selected forhumanization, an appropriate human acceptor binding molecule may beobtained, e.g., from sequence databases of expressed human antibodygenes, from germline Ig sequences or a consensus sequence of severalhuman binding molecules.

In one embodiment, a CDR homology based method is used for humanization(see, e.g., Hwang, W. Y. K., et al. (2005) Methods 36:35, the contentsof which is incorporated in its entirety herein by this reference). Thismethod generally involves substitution of mouse CDRs into a humanvariable domain framework based on similarly structured mouse and humanCDRs rather than similarly structured mouse and human frameworks. Thesimilarity of the mouse and human CDRs is generally determined byidentifying human genes of the same chain type (light or heavy) thathave the same combination of canonical CDR structures as the mousebinding molecules and thus retain three-dimensional conformation of CDRpeptide backbones. Secondly, for each of the candidate variable geneswith matching canonical structures, residue to residue homology betweenthe mouse and candidate human CDRs is evaluated. Finally, to generate ahumanized binding molecule, CDR residues of the chosen human candidateCDR not already identical to the mouse CDR are converted to the mousesequence. In one embodiment, no mutations of the human framework areintroduced into the humanized binding molecule.

In one embodiment, human germline sequences are evaluated for CDRhomology to the GITR binding molecule CDRs. For example, for the murine6C8 antibody, all germ line light chain kappa chain V genes with a 2-1-1canonical structure in the IMGT database were compared with the 6C8antibody sequence. The same was done for the heavy chain where all 3-1germ line heavy chain V genes were compared to the 6C8 amino acidsequence. Accordingly, in one embodiment, a binding molecule of theinvention comprises a human kappa chain V region framework with a 2-1-1canonical structure. In another embodiment, a binding molecule of theinvention comprises a human heavy chain V region framework with a 3-1canonical structure.

The following potential human light chain germline sequences wereidentified and may be incorporated into a binding molecule of theinvention:

The IMGT accession number of the IGKV3-15 gene is M23090. The amino acidsequence is:

(SEQ ID NO: 25) EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP.

The IMGT accession number of the IGKV3D-11 gene is X17264. The aminoacid sequence is:

(SEQ ID NO: 26) EIVLTQSPATLSLSPGERATLSCRASQGVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRSNWH.

There are two alleles of the IGKV3-11 gene. The IMGT accession number ofallele *01 of the IGKV3-11gene is X01668. The amino acid sequence is:

(SEQ ID NO: 27) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWP.

The IMGT accession number of allele *02 of the IGKV3-11gene is K02768.The amino acid sequence is:

(SEQ ID NO: 28) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGRDFTLTISSLEPEDFAVYYCQQRSNWP.

The IMGT accession number of the IGKV1D-43 gene is X72817. The aminoacid sequence is:

(SEQ ID NO: 29) AIRMTQSPFSLSASVGDRVTITCWASQGISSYLAWYQQKPAKAPKLFIYYASSLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYYSTP.

There are two alleles of the IGKV1-39 gene. The IMGT accession number ofallele *01 of the IGKV1-39 gene is X59315. The amino acid sequence is:

(SEQ ID NO: 30) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP.

The IMGT accession number of allele *02 of the IGKV1-39 gene is X59318.The amino acid sequence is:

(SEQ ID NO: 31) DIQMTQSPSFLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQCGYSTP.

The IMGT accession number of the IGKV1-33 gene is M64856. The amino acidsequence is:

(SEQ ID NO: 32) DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLP.

The IMGT accession number of the IGKV1-27 gene is X63398. The amino acidsequence is:

(SEQ ID NO: 33) DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNSAP.

There are two alleles of the IGKV1-17 gene. The IMGT accession number ofallele *01 of the IGKV1-17 gene is X72808. The amino acid sequence is:

(SEQ ID NO: 34) DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCLQHNSYP.

The IMGT accession number of allele *02 of the IGKV1-17 gene is D88255.The amino acid sequence is:

(SEQ ID NO: 35) DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISNLQPEDFATYYCLQHNSYP.

There are two alleles of the IGKV1D-16 gene. The IMGT accession numberof allele *01 of the IGKV1D-16 gene is K01323. The amino acid sequenceis:

(SEQ ID NO: 36) DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP.

The IMGT accession number of allele *02 of the IGKV1D-16 gene is J00244.The amino acid sequence is:

(SEQ ID NO: 37) DIQMTQSPSSLSASVGDRVTITCRARQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP.

The IMGT accession number of the IGKV1-16 gene is J00248. The amino acidsequence is:

(SEQ ID NO: 38) DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWFQQKPGKAPKSLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYP.

There are two alleles of the IGKV1-12 gene. The IMGT accession number ofallele *01 of the IGKV1-12 gene is V01577. The amino acid sequence is:

(SEQ ID NO: 39) DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFP.

The IMGT accession number of allele *02 of the IGKV1-12 gene is V01576.The amino acid sequence is:

(SEQ ID NO: 40) DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFP.

The IMGT accession number of the IGKV1-9 gene is Z00013. The amino acidsequence is:

(SEQ ID NO: 41) DIQLTQSPSFLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQLNSYP.

The IMGT accession number of the IGKV1-6 gene is M64858. The amino acidsequence is:

(SEQ ID NO: 42) AIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQDYNYP.

There are three alleles of the IGKV1-5 gene. The IMGT accession numberof allele *01 of the IGKV1-5 gene is Z00001. The amino acid sequence is:

(SEQ ID NO: 43) DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPKLLIYDASSLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYS.

The following potential human heavy chain germline sequences wereidentified and may be incorporated into a binding molecule of theinvention:

There are ten alleles of the IGHV2-5 gene. The IMGT accession number ofallele *01 of the IGHV2-5 gene is X62111. The amino acid sequence is:

(SEQ ID NO: 45) QITLKESGPTLVKPTQTLTLTCTFSGFSLSTSGVGVGWIRQPPGKALEWLALIYWNDDKRYSPSLKSRLTITKDTSKNQVVLTMTNMDPVDTATYY.

The IMGT accession number of the IGHV2-26 gene is M99648. The amino acidsequence is:

(SEQ ID NO: 46) QVTLKESGPVLVKPTETLTLTCTVSGFSLSNARMGVSWIRQPPGKALEWLAHIFSNDEKSYSTSLKSRLTISKDTSKSQVVLTMTNMDPVDTATYYCA RI.

There are thirteen alleles of the IGHV2-70 gene. The IMGT accessionnumber of allele *01 of the IGHV2-70 gene is L21969. The amino acidsequence is:

(SEQ ID NO: 47) QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMCVSWIRQPPGKALEWLALIDWDDDKYYSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYYCA RI.

There are four alleles of the IGHV4-30-2 gene. The IMGT accession numberof allele *01 of the IGHV4-30-2 gene is L10089. The amino acid sequenceis:

(SEQ ID NO: 48) QLQLQESGSGLVKPSQTLSLTCAVSGGSISSGGYSWSWIRQPPGKGLEWIGYIYHSGSTYYNPSLKSRVTISVDRSKNQFSLKLSSVTAADTAVYYC AR.

There are six alleles of the IGHV4-30-4 gene. The IMGT accession numberof allele *01 of the IGHV4-30-4 gene is Z14238. The amino acid sequenceis:

(SEQ ID NO: 49) QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGDYYWSWIRQPPGKGLEWIGYIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC AR.

There are ten alleles of the IGHV4-31 gene. The IMGT accession number ofallele *01 of the IGHV2-5 gene is L10098. The amino acid sequence is:

(SEQ ID NO: 50) QVQLQESGPGLVKPSQTLSLTCTVSGGSISSGGYYWSWIRQHPGKGLEWIGYIYYSGSTYYNPSLKSLVTISVDTSKNQFSLKLSSVTAADTAVYYC AR.

There are six alleles of the IGHV4-39 gene. The IMGT accession number ofallele *01 of the IGHV4-39 gene is L10094. The amino acid sequence is:

(SEQ ID NO: 51) QLQLQESGPGLVKPSETLSLTCTVSGGSISSSSYYWGWIRQPPGKGLEWIGSIYYSGSTYYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC AR.

There are eight alleles of the IGHV4-61 gene. The IMGT accession numberof allele *01 of the IGHV4-61 gene is M29811. The amino acid sequenceis:

(SEQ ID NO: 52) QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYC AR.

Each of these germline sequences may be used to provide frameworkregions for use with one or more 6C8 CDRs.

As used herein, “canonical structures” are conserved hypervariable loopconformations made by different CDRs by which the binding molecule formsthe antigen contacts. The assignment of canonical structure classes to anew binding molecule can be achieved using publicly available software.

In another embodiment, the substitution of mouse CDRs into a humanvariable domain framework is based on the retention of the correctspatial orientation of the mouse variable domain framework byidentifying human variable domain frameworks which will retain the sameconformation as the mouse variable domain frameworks from which the CDRswere derived. In one embodiment, this is achieved by obtaining the humanvariable domains from human binding molecules whose framework sequencesexhibit a high degree of sequence identity with the murine variableframework domains from which the CDRs were derived. See Kettleborough etal., Protein Engineering 4:773 (1991); Kolbinger et al., ProteinEngineering 6:971 (1993) and Carter et al., WO 92/22653.

Preferably the human acceptor binding molecule retains the canonical andinterface residues of the donor binding molecule. Additionally, thehuman acceptor binding molecule preferably has substantial similarity inthe length of CDR loops. See Kettleborough et al., Protein Engineering4:773 (1991); Kolbinger et al., Protein Engineering 6:971 (1993) andCarter et al., WO 92/22653.

In another embodiment, appropriate human acceptor sequences may beselected based on homology to framework regions of the 6C8 bindingmolecule. For example, the amino acid sequence of the 6C8 bindingmolecule may be compared to the amino acid sequence of other knownbinding molecules by, for example, by comparing the FR regions or thevariable region sequences of the 6C8 amino acid sequence against apubicly available database of known binding molecules and selectingthose sequences with the highest percent identity of amino acids in thevariable or FR region, i.e., 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%. In one embodiment, theframework sequence set forth in SEQ ID NO:67 may be used

GFSLSTSGMGVG

HIWWD DDKYNPSLKS

ARTRRYFPFAY

(SEQ ID NO:67; Framework residues are in bold)). In another embodiment,the framework sequence set forth in SEQ ID NO:68 may be used

GFSLSTSGMGVG

HIWW DDDKYNPSLK

ARTRRYFPFAY

(SEQ ID NO:68; Framework residues are in bold)).

Having identified the complementarity determining regions of the murinedonor immunoglobulin and appropriate human acceptor immunoglobulins, thenext step is to determine which, if any, residues from these componentsshould be substituted to optimize the properties of the resultinghumanized binding molecule. In general, substitution of human amino acidresidues with murine should be minimized, because introduction of murineresidues increases the risk of the binding molecule eliciting ahuman-anti-mouse-antibody (HAMA) response in humans. Art-recognizedmethods of determining immune response can be performed to monitor aHAMA response in a particular patient or during clinical trials.Patients administered humanized binding molecules can be given animmunogenicity assessment at the beginning and throughout theadministration of said therapy. The HAMA response is measured, forexample, by detecting antibodies to the humanized therapeutic reagent,in serum samples from the patient using a method known to one in theart, including surface plasmon resonance technology (BIACORE) and/orsolid-phase ELISA analysis.

When necessary, one or more residues in the human framework regions canbe changed or substituted to residues at the corresponding positions inthe murine antibody so as to preserve the binding affinity of thehumanized antibody to the antigen. This change is sometimes called“backmutation”. Certain amino acids from the human variable regionframework residues are selected for back mutation based on theirpossible influence on CDR conformation and/or binding to antigen. Theplacement of murine CDR regions with human variable framework region canresult in conformational restraints, which, unless corrected bysubstitution of certain amino acid residues, lead to loss of bindingaffinity.

In one embodiment, the selection of amino acid residues for backmutationcan be determined, in part, by computer modeling, using art recognizedtechniques. In general, molecular models are produced starting fromsolved structures for immunoglobulin chains or domains thereof. Thechains to be modeled are compared for amino acid sequence similaritywith chains or domains of solved three-dimensional structures, and thechains or domains showing the greatest sequence similarity is/areselected as starting points for construction of the molecular model.Chains or domains sharing at least 50% sequence identity are selectedfor modeling, and preferably those sharing at least 60%, 70%, 80%, 90%sequence identity or more are selected for modeling. The solved startingstructures are modified to allow for differences between the actualamino acids in the immunoglobulin chains or domains being modeled, andthose in the starting structure. The modified structures are thenassembled into a composite immunoglobulin. Finally, the model is refinedby energy minimization and by verifying that all atoms are withinappropriate distances from one another and that bond lengths and anglesare within chemically acceptable limits.

The selection of amino acid residues for substitution can also bedetermined, in part, by examination of the characteristics of the aminoacids at particular locations, or empirical observation of the effectsof substitution or mutagenesis of particular amino acids. For example,when an amino acid differs between a murine variable region frameworkresidue and a selected human variable region framework residue, thehuman framework amino acid may be substituted by the equivalentframework amino acid from the mouse binding molecule when it isreasonably expected that the amino acid: (1) noncovalently binds antigendirectly, (2) is adjacent to a CDR region, (3) otherwise interacts witha CDR region (e.g., is within about 3-6 Å of a CDR region as determinedby computer modeling), or (4) participates in the VL-VH interface.

Residues which “noncovalently bind antigen directly” include amino acidsin positions in framework regions which are have a good probability ofdirectly interacting with amino acids on the antigen according toestablished chemical forces, for example, by hydrogen bonding, Van derWaals forces, hydrophobic interactions, and the like.

Residues which are “adjacent to a CDR region” include amino acidresidues in positions immediately adjacent to one or more of the CDRs inthe primary sequence of the humanized immunoglobulin chain, for example,in positions immediately adjacent to a CDR as defined by Kabat, or a CDRas defined by Chothia (See e.g., Chothia and Lesk JMB 196:901 (1987)).These amino acids are particularly likely to interact with the aminoacids in the CDRs and, if chosen from the acceptor, may distort thedonor CDRs and reduce affinity. Moreover, the adjacent amino acids mayinteract directly with the antigen (Amit et al., Science, 233:747(1986), which is incorporated herein by reference) and selecting theseamino acids from the donor may be desirable to keep all the antigencontacts that provide affinity in the original binding molecule.

Residues that “otherwise interact with a CDR region” include those thatare determined by secondary structural analysis to be in a spatialorientation sufficient to effect a CDR region. In one embodiment,residues that “otherwise interact with a CDR region” are identified byanalyzing a three-dimensional model of the donor immunoglobulin (e.g., acomputer-generated model). A three-dimensional model, typically of theoriginal donor binding molecule, shows that certain amino acids outsideof the CDRs are close to the CDRs and have a good probability ofinteracting with amino acids in the CDRs by hydrogen bonding, Van derWaals forces, hydrophobic interactions, etc. At those amino acidpositions, the donor immunoglobulin amino acid rather than the acceptorimmunoglobulin amino acid may be selected. Amino acids according to thiscriterion will generally have a side chain atom within about 3 Å of someatom in the CDRs and must contain an atom that could interact with theCDR atoms according to established chemical forces, such as those listedabove.

In the case of atoms that may form a hydrogen bond, the 3 Å is measuredbetween their nuclei, but for atoms that do not form a bond, the 3 Å ismeasured between their Van der Waals surfaces. Hence, in the lattercase, the nuclei must be within about 6 Å (3 Å plus the sum of the Vander Waals radii) for the atoms to be considered capable of interacting.In many cases the nuclei will be from 4 or 5 to 6 Å apart. Indetermining whether an amino acid can interact with the CDRs, it ispreferred not to consider the last 8 amino acids of heavy chain CDR aspart of the CDRs, because from the viewpoint of structure, these 8 aminoacids behave more as part of the framework.

Amino acids that are capable of interacting with amino acids in theCDRs, may be identified in yet another way. The solvent accessiblesurface area of each framework amino acid is calculated in two ways: (1)in the intact binding molecule, and (2) in a hypothetical moleculeconsisting of the binding molecule with its CDRs removed. A significantdifference between these numbers of about 10 square angstroms or moreshows that access of the framework amino acid to solvent is at leastpartly blocked by the CDRs, and therefore that the amino acid is makingcontact with the CDRs. Solvent accessible surface area of an amino acidmay be calculated based on a three-dimensional model of an bindingmolecule, using algorithms known in the art (e.g., Connolly, J. Appl.Cryst. 16:548 (1983) and Lee and Richards, J. Mol. Biol. 55:379 (1971),both of which are incorporated herein by reference). Framework aminoacids may also occasionally interact with the CDRs indirectly, byaffecting the conformation of another framework amino acid that in turncontacts the CDRs.

The amino acids at several positions in the framework are known to becapable of interacting with the CDRs in many binding molecules (Chothiaand Lesk, supra, Chothia et al., supra and Tramontano et al., J. Mol.Biol. 215:175 (1990), all of which are incorporated herein byreference). Notably, the amino acids at positions 2, 48, 64 and 71 ofthe light chain and 26-30, 71 and 94 of the heavy chain (numberingaccording to Kabat) are known to be capable of interacting with the CDRsin many binding molecules. The amino acids at positions 35 in the lightchain and 93 and 103 in the heavy chain are also likely to interact withthe CDRs. At all these numbered positions, choice of the donor aminoacid rather than the acceptor amino acid (when they differ) to be in thehumanized immunoglobulin is preferred. On the other hand, certainresidues capable of interacting with the CDR region, such as the first 5amino acids of the light chain, may sometimes be chosen from theacceptor immunoglobulin without loss of affinity in the humanizedbinding molecule.

Residues which “participate in the VL-VH interface” or “packingresidues” include those residues at the interface between VL and VH asdefined, for example, by Novotny and Haber (Proc. Natl. Acad. Sci. USA,82:4592-66 (1985)) or Chothia et al, supra. Generally, unusual packingresidues should be retained in the humanized binding molecule if theydiffer from those in the human frameworks.

In general, one or more of the amino acids fulfilling the above criteriais substituted. In some embodiments, all or most of the amino acidsfulfilling the above criteria are substituted. Occasionally, there issome ambiguity about whether a particular amino acid meets the abovecriteria, and alternative variant binding molecules are produced, one ofwhich has that particular substitution, the other of which does not.Alternative variant binding molecules so produced can be tested in anyof the assays described herein for the desired activity, and thepreferred binding molecule selected.

Usually the CDR regions in humanized binding molecules are substantiallyidentical, and more usually, identical to the corresponding CDR regionsof the donor binding molecule. Although not usually desirable, it issometimes possible to make one or more conservative amino acidsubstitutions of CDR residues without appreciably affecting the bindingaffinity of the resulting humanized binding molecule. By conservativesubstitutions it is meant combinations such as Gly, Ala; Val, Ile, Leu;Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.

Additional candidates for substitution are acceptor human frameworkamino acids that are unusual or “rare” for a human immunoglobulin atthat position. These amino acids can be substituted with amino acidsfrom the equivalent position of the mouse donor binding molecule or fromthe equivalent positions of more typical human immunoglobulins. Forexample, substitution may be desirable when the amino acid in a humanframework region of the acceptor immunoglobulin is rare for thatposition and the corresponding amino acid in the donor immunoglobulin iscommon for that position in human immunoglobulin sequences; or when theamino acid in the acceptor immunoglobulin is rare for that position andthe corresponding amino acid in the donor immunoglobulin is also rare,relative to other human sequences. These criterion help ensure that anatypical amino acid in the human framework does not disrupt the bindingmolecule structure. Moreover, by replacing an unusual human acceptoramino acid with an amino acid from the donor binding molecule thathappens to be typical for human binding molecules, the humanized bindingmolecule may be made less immunogenic.

The term “rare”, as used herein, indicates an amino acid occurring atthat position in less than about 20% but usually less than about 10% ofsequences in a representative sample of sequences, and the term“common”, as used herein, indicates an amino acid occurring in more thanabout 25% but usually more than about 50% of sequences in arepresentative sample. For example, all human light and heavy chainvariable region sequences are respectively grouped into “subgroups” ofsequences that are especially homologous to each other and have the sameamino acids at certain critical positions (Kabat et al., supra). Whendeciding whether an amino acid in a human acceptor sequence is “rare” or“common” among human sequences, it will often be preferable to consideronly those human sequences in the same subgroup as the acceptorsequence.

Additional candidates for substitution are acceptor human frameworkamino acids that would be identified as part of a CDR region under thealternative definition proposed by Chothia et al., supra. Additionalcandidates for substitution are acceptor human framework amino acidsthat would be identified as part of a CDR region under the AbM and/orcontact definitions. Notably, CDR1 in the variable heavy chain isdefined as including residues 26-32.

Additional candidates for substitution are acceptor framework residuesthat correspond to a rare or unusual donor framework residue. Rare orunusual donor framework residues are those that are rare or unusual (asdefined herein) for murine binding molecules at that position. Formurine binding molecules, the subgroup can be determined according toKabat and residue positions identified which differ from the consensus.These donor specific differences may point to somatic mutations in themurine sequence which enhances activity. Unusual residues that arepredicted to affect binding are retained, whereas residues predicted tobe unimportant for binding can be substituted.

Additional candidates for substitution are non-germline residuesoccurring in an acceptor framework region. For example, when an acceptorbinding molecule chain (i.e., a human binding molecule chain sharingsignificant sequence identity with the donor binding molecule chain) isaligned to a germline binding molecule chain (likewise sharingsignificant sequence identity with the donor chain), residues notmatching between acceptor chain framework and the germline chainframework can be substituted with corresponding residues from thegermline sequence.

Other than the specific amino acid substitutions discussed above, theframework regions of humanized binding molecules are usuallysubstantially identical, and more usually, identical to the frameworkregions of the human binding molecules from which they were derived. Ofcourse, many of the amino acids in the framework region make little orno direct contribution to the specificity or affinity of a bindingmolecule. Thus, many individual conservative substitutions of frameworkresidues can be tolerated without appreciable change of the specificityor affinity of the resulting humanized binding molecule. Thus, in oneembodiment the variable framework region of the humanized bindingmolecule shares at least 85% sequence identity to a human variableframework region sequence or consensus of such sequences. In anotherembodiment, the variable framework region of the humanized bindingmolecule shares at least 90%, preferably 95%, more preferably 96%, 97%,98% or 99% sequence identity to a human variable framework regionsequence or consensus of such sequences. In general, however, suchsubstitutions are undesirable.

In one embodiment, a binding molecule of the invention further comprisesat least one backmutation of a human amino acid residue to thecorresponding mouse amino acid residue where the amino acid residue isan interface packing residue. “Interface packing residues” include thoseresidues at the interface between VL and VH as defined, for example, byNovotny and Haber, Proc. Natl. Acad. Sci. USA, 82:4592-66 (1985).

In one embodiment, a binding molecule of the invention further comprisesat least one backmutation of a human amino acid residue to thecorresponding mouse amino acid residue is a canonical residue.“Canonical residues” are conserved framework residues within a canonicalor structural class known to be important for CDR conformation(Tramontano et al., J. Mol. Biol. 215:175 (1990), all of which areincorporated herein by reference). Canonical residues include 2, 25,27B, 28, 29, 30, 33, 48, 51, 52, 64, 71, 90, 94 and 95 of the lightchain and residues 24, 26, 27 29, 34, 54, 55, 71 and 94 of the heavychain. Additional residues (e.g., CDR structure-determining residues)can be identified according to the methodology of Martin and Thorton(1996) J. Mol. Biol. 263:800.

In one embodiment, a binding molecule of the invention further comprisesat least one backmutation of a human amino acid residue to thecorresponding mouse amino acid residue where the amino acid residue isat a position capable of interacting with a CDR. Notably, the aminoacids at positions 2, 48, 64 and 71 of the light chain and 26-30, 71 and94 of the heavy chain (numbering according to Kabat) are known to becapable of interacting with the CDRs in many antibodies. The amino acidsat positions 35 in the light chain and 93 and 103 in the heavy chain arealso likely to interact with the CDRs.

Based on CLUSTAL W analysis, several amino acid residues in the humanframework were identified for potential substitution, e.g., withcorresponding amino acid residues from the 6C8 light chain. Theseincluded positions 1, 8, 9, 10, 11, 13, 15, 17, 19, 20, 21, 22, 43, 45,46, 58, 60, 63, 70, 76, 77, 78, 79, 83, 85, 87, 100, and 104.

In one embodiment, a variable light chain framework of a bindingmolecule of the invention further comprises at least one substitution ofa human amino acid residue to the corresponding mouse amino acid residueselected from the group consisting of: E1D (i.e., the E at position 1 ofthe CDR-grafted antibody which comprises murine CDRs and human FRregaions is mutated to a D, which is the corresponding amino acidresidue in the 6C8 antibody), P8Q, A9K, T10F, L11M, V13T, P15V, E17D,A19V, T20S, L21V, S22T, A43S, R45K, L46A, I58V, A60D, S63T, E70D, S76N,S77N, L78V, Q79H, F83L, V85E, Y87F, G100A, and V104L.

Based on CLUSTAL W analysis, several amino acid residues in the humanframework were identified for potential substitution, e.g., withcorresponding amino acid residues from the 6C8 heavy chain. Theseincluded positions 5, 10, 11, 12, 15, 19, 23, 43, 46, 68, 77, 81, 83,84, 86, 87, 89, 90, and 92.

In one embodiment, a variable heavy chain framework of a bindingmolecule of the invention further comprises at least one substitution ofa human amino acid residue to the corresponding mouse amino acid residueselected from the group consisting of: R5K (i.e., the R at position 5 ofthe CDR-grafted antibody which comprises murine CDRs and human FRregaions is mutated to a K, which is the corresponding amino acidresidue in the 6C8 antibody), A10G, L11I, V12L, T15S, T19S, T23S, P43S,A46G, R68Q, K77R, V81F, T83K, M84I, N86S, M87V, P89T, V90A, and T92A.

The humanized binding molecules preferably exhibit a specific bindingaffinity for antigen of at least 10⁷, 10⁸, 10⁹ or 10¹⁰ M⁻¹. Usually theupper limit of binding affinity of the humanized binding molecules forantigen is within a factor of three, four or five of that of the donorimmunoglobulin. Often the lower limit of binding affinity is also withina factor of three, four or five of that of donor immunoglobulin.Alternatively, the binding affinity can be compared to that of ahumanized binding molecule having no substitutions (e.g., a bindingmolecule having donor CDRs and acceptor FRs, but no FR substitutions).In such instances, the binding of the optimized binding molecule (withsubstitutions) is preferably at least two- to three-fold greater, orthree- to four-fold greater, than that of the unsubstituted bindingmolecule. For making comparisons, activity of the various bindingmolecules can be determined, for example, by BIACORE (i.e., surfaceplasmon resonance using unlabelled reagents) or competitive bindingassays.

Having conceptually selected the CDR and framework components ofhumanized binding molecules, a variety of methods are available forproducing such binding molecules. Because of the degeneracy of the code,a variety of nucleic acid sequences will encode each binding moleculeamino acid sequence. The desired nucleic acid sequences can be producedby de novo solid-phase DNA synthesis or by PCR mutagenesis of an earlierprepared variant of the desired polynucleotide. Oligonucleotide-mediatedmutagenesis is a preferred method for preparing substitution, deletionand insertion variants of target polypeptide DNA. See Adelman et al.(DNA 2:183 (1983)). Briefly, the target polypeptide DNA is altered byhybridizing an oligonucleotide encoding the desired mutation to asingle-stranded DNA template. After hybridization, a DNA polymerase isused to synthesize an entire second complementary strand of the templatethat incorporates the oligonucleotide primer, and encodes the selectedalteration in the target polypeptide DNA.

The variable segments of binding molecules produced as described supra(e.g., the heavy and light chain variable regions of chimeric,humanized, or human binding molecules) are typically linked to at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin. Human constant region DNA sequences can beisolated in accordance with well known procedures from a variety ofhuman cells, but preferably immortalized B cells (see Kabat et al.,supra, and Liu et al., WO87/02671) (each of which is incorporated byreference in its entirety for all purposes). Ordinarily, the bindingmolecule will contain both light chain and heavy chain constant regions.The heavy chain constant region usually includes CH1, hinge, CH2, CH3,and CH4 regions. A binding molecule described herein include antibodieshaving all types of constant regions, including IgM, IgG, IgD, IgA andIgE, and any isotype, including IgGl, IgG2, IgG3 and IgG4. The choice ofconstant region depends, in part, or whether binding molecule-dependentcomplement and/or cellular mediated toxicity is desired. For example,isotopes IgG1 and IgG3 have complement activity and isotypes IgG2 andIgG4 do not. When it is desired that the binding molecule (e.g.,humanized binding molecule) exhibit cytotoxic activity, the constantdomain is usually a complement fixing constant domain and the class istypically IgG1. When such cytotoxic activity is not desirable, theconstant domain may be, e.g., of the IgG2 class. Choice of isotype canalso affect passage of antibody into the brain. Human isotype IgG1 ispreferred. Light chain constant regions can be lambda or kappa. Thehumanized binding molecule may comprise sequences from more than oneclass or isotype. Binding molecules can be expressed as tetramerscontaining two light and two heavy chains, as separate heavy chains,light chains, as Fab, Fab′ F(ab′)2, and Fv, or as single chain bindingmolecules in which heavy and light chain variable domains are linkedthrough a spacer.

III. Production of Binding Molecules

The present invention features binding molecules having specificity forGITR, e.g., human GITR. Such binding molecules can be used informulating various therapeutic compositions of the invention or,preferably, provide complementarity determining regions for theproduction of humanized or chimeric binding molecules (described indetail below). The production of non-human monoclonal binding molecules,e.g., murine, guinea pig, primate, rabbit or rat, can be accomplishedby, for example, immunizing the animal with GITR or with a nucleic acidmolecule encoding GITR. For example, the 6C8 binding molecule was madeby placing the gene encoding human GITR in an expression vector andimmunizing animals. A longer polypeptide comprising GITR or animmunogenic fragment of GITR or anti-idiotypic binding molecule of GITRcan also be used. (see, for example, Harlow & Lane, supra, incorporatedby reference for all purposes). Such an immunogen can be obtained from anatural source, by peptide synthesis or by recombinant expression.Optionally, the immunogen can be administered, fused or otherwisecomplexed with a carrier protein, as described below. Optionally, theimmunogen can be administered with an adjuvant. The term “adjuvant”refers to a compound that when administered in conjunction with anantigen augments the immune response to the antigen, but whenadministered alone does not generate an immune response to the antigen.Adjuvants can augment an immune response by several mechanisms includinglymphocyte recruitment, stimulation of B and/or T cells, and stimulationof macrophages. Several types of adjuvants can be used as describedbelow. Complete Freund's adjuvant followed by incomplete adjuvant ispreferred for immunization of laboratory animals.

Rabbits or guinea pigs are typically used for making polyclonal bindingmolecules. Exemplary preparation of polyclonal binding molecules, e.g.,for passive protection, can be performed as follows. Animals areimmunized with 100 μg GITR, plus adjuvant, and euthanized at 4-5 months.Blood is collected and IgG is separated from other blood components.Binding molecules specific for the immunogen may be partially purifiedby affinity chromatography. An average of about 0.5-1.0 mg ofimmunogen-specific binding molecule is obtained per animal, giving atotal of 60-120 mg.

Mice are typically used for making monoclonal binding molecules.Monoclonals can be prepared against a fragment by injecting the fragmentor longer form of GITR into a mouse, preparing hybridomas and screeningthe hybridomas for a binding molecule that specifically binds to GITR.Optionally, binding molecules are screened for binding to a specificregion or desired fragment of GITR without binding to othernonoverlapping fragments of GITR. The latter screening can beaccomplished by determining binding of a binding molecule to acollection of deletion mutants of a GITR peptide and determining whichdeletion mutants bind to the binding molecule. Binding can be assessed,for example, by Western blot or ELISA. The smallest fragment to showspecific binding to the binding molecule defines the epitope of thebinding molecule. Alternatively, epitope specificity can be determinedby a competition assay in which a test and reference binding moleculecompete for binding to GITR. If the test and reference binding moleculecompete, then they bind to the same epitope (or epitopes sufficientlyproximal) such that binding of one binding molecule interferes withbinding of the other. The preferred isotype for such binding moleculesis mouse isotype IgG2a or equivalent isotype in other species. Mouseisotype IgG2a is the equivalent of human isotype IgG1.

In another embodiment, DNA encoding a binding molecule may be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine binding molecules). Theisolated and subcloned hybridoma cells serve as a preferred source ofsuch DNA. Once isolated, the DNA may be placed into expression vectors,which are then transfected into prokaryotic or eukaryotic host cellssuch as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO)cells or myeloma cells that do not otherwise produce immunoglobulins.More particularly, the isolated DNA (which may be synthetic as describedherein) may be used to clone constant and variable region sequences forthe manufacture of binding molecules as described in Newman et al., U.S.Pat. No. 5,658,570, filed Jan. 25, 1995, which is incorporated byreference herein. Essentially, this entails extraction of RNA from theselected cells, conversion to cDNA, and amplification by PCR using Igspecific primers. Suitable primers for this purpose are also describedin U.S. Pat. No. 5,658,570. Transformed cells expressing the desiredantibody may be produced in relatively large quantities to provideclinical and commercial supplies of the binding molecule.

Those skilled in the art will also appreciate that DNA encoding bindingmolecules or fragments thereof (e.g., antigen binding sites) may also bederived from antibody phage libraries, e.g., using pd phage or Fdphagemid technology. Exemplary methods are set forth, for example, in EP368 684 B1; U.S. Pat. No. 5,969,108, Hoogenboom, H. R. and Chames. 2000.Immunol. Today 21:371; Nagy et al. 2002. Nat. Med. 8:801; Huie et al.2001. Proc. Natl. Acad. Sci. USA 98:2682; Lui et al. 2002. J. Mol. Biol.315:1063, each of which is incorporated herein by reference. Severalpublications (e.g., Marks et al. Bio/Technology 10:779-783 (1992)) havedescribed the production of high affinity human binding molecules bychain shuffling, as well as combinatorial infection and in vivorecombination as a strategy for constructing large phage libraries. Inanother embodiment, Ribosomal display can be used to replacebacteriophage as the display platform (see, e.g., Hanes et al. 2000.Nat. Biotechnol. 18:1287; Wilson et al. 2001. Proc. Natl. Acad. Sci. USA98:3750; or Irving et al. 2001 J. Immunol. Methods 248:31. In yetanother embodiment, cell surface libraries can be screened for bindingmolecules (Boder et al. 2000. Proc. Natl. Acad. Sci. USA 97:10701;Daugherty et al. 2000 J. Immunol. Methods 243:211. Such proceduresprovide alternatives to traditional hybridoma techniques for theisolation and subsequent cloning of monoclonal binding molecules.

Yet other embodiments of the present invention comprise the generationof human or substantially human binding molecules in transgenic animals(e.g., mice) that are incapable of endogenous immunoglobulin production(see e.g., U.S. Pat. Nos. 6,075,181, 5,939,598, 5,591,669 and 5,589,369each of which is incorporated herein by reference). For example, it hasbeen described that the homozygous deletion of the antibody heavy-chainjoining region in chimeric and germ-line mutant mice results in completeinhibition of endogenous antibody production. Transfer of a humanimmunoglobulin gene array to such germ line mutant mice will result inthe production of human binding molecules upon antigen challenge.Another preferred means of generating human binding molecules using SCIDmice is disclosed in U.S. Pat. No. 5,811,524 which is incorporatedherein by reference. It will be appreciated that the genetic materialassociated with these human binding molecules may also be isolated andmanipulated as described herein.

Yet another highly efficient means for generating recombinant bindingmolecules is disclosed by Newman, Biotechnology, 10: 1455-1460 (1992).Specifically, this technique results in the generation of primatizedbinding molecules that contain monkey variable domains and humanconstant sequences. This reference is incorporated by reference in itsentirety herein. Moreover, this technique is also described in U.S. Pat.Nos. 5,658,570, 5,693,780 and 5,756,096 each of which is incorporatedherein by reference.

In another embodiment, lymphocytes can be selected by micromanipulationand the variable genes isolated. For example, peripheral bloodmononuclear cells can be isolated from an immunized mammal and culturedfor about 7 days in vitro. The cultures can be screened for specificIgGs that meet the screening criteria. Cells from positive wells can beisolated. Individual Ig-producing B cells can be isolated by FACS or byidentifying them in a complement-mediated hemolytic plaque assay.Ig-producing B cells can be micromanipulated into a tube and the VH andVL genes can be amplified using, e.g., RT-PCR. The VH and VL genes canbe cloned into an antibody expression vector and transfected into cells(e.g., eukaryotic or prokaryotic cells) for expression.

Moreover, genetic sequences useful for producing the polypeptides of thepresent invention may be obtained from a number of different sources.For example, as discussed extensively above, a variety of human antibodygenes are available in the form of publicly accessible deposits. Manysequences of antibodies and antibody-encoding genes have been publishedand suitable antibody genes can be chemically synthesized from thesesequences using art recognized techniques. Oligonucleotide synthesistechniques compatible with this aspect of the invention are well knownto the skilled artisan and may be carried out using any of severalcommercially available automated synthesizers. In addition, DNAsequences encoding several types of heavy and light chains set forthherein can be obtained through the services of commercial DNA synthesisvendors. The genetic material obtained using any of the foregoingmethods may then be altered or synthetic to provide obtain polypeptidesof the present invention.

Alternatively, antibody-producing cell lines may be selected andcultured using techniques well known to the skilled artisan. Suchtechniques are described in a variety of laboratory manuals and primarypublications. In this respect, techniques suitable for use in theinvention as described below are described in Current Protocols inImmunology, Coligan et al., Eds., Green Publishing Associates andWiley-Interscience, John Wiley and Sons, New York (1991) which is hereinincorporated by reference in its entirety, including supplements.

As is well known, RNA may be isolated from the original hybridoma cellsor from other transformed cells by standard techniques, such asguanidinium isothiocyanate extraction and precipitation followed bycentrifugation or chromatography. Where desirable, mRNA may be isolatedfrom total RNA by standard techniques such as chromatography on oligo dTcellulose. Suitable techniques are familiar in the art.

In one embodiment, cDNAs that encode the light and the heavy chains ofthe binding molecule may be made, either simultaneously or separately,using reverse transcriptase and DNA polymerase in accordance with wellknown methods. PCR may be initiated by consensus constant region primersor by more specific primers based on the published heavy and light chainDNA and amino acid sequences. As discussed above, PCR also may be usedto isolate DNA clones encoding the binding molecule light and heavychains. In this case the libraries may be screened by consensus primersor larger homologous probes, such as mouse constant region probes.

DNA, typically plasmid DNA, may be isolated from the cells usingtechniques known in the art, restriction mapped and sequenced inaccordance with standard, well known techniques set forth in detail,e.g., in the foregoing references relating to recombinant DNAtechniques. Of course, the DNA may be synthetic according to the presentinvention at any point during the isolation process or subsequentanalysis.

In one embodiment, a binding molecule of the invention comprises orconsists of an antigen binding fragment of an antibody. The term“antigen-binding fragment” refers to a polypeptide fragment of animmunoglobulin or antibody that binds antigen or competes with intactantibody (i.e., with the intact antibody from which they were derived)for antigen binding (i.e., specific binding). As used herein, the term“fragment” of an antibody molecule includes antigen-binding fragments ofantibodies, for example, an antibody light chain (VL), an antibody heavychain (VH), a single chain antibody (scFv), a F(ab′)2 fragment, a Fabfragment, an Fd fragment, an Fv fragment, and a single domain antibodyfragment (DAb). Fragments can be obtained, e.g., via chemical orenzymatic treatment of an intact or complete antibody or antibody chainor by recombinant means.

In one embodiment, a binding molecule of the invention is an engineeredor modified antibody. Engineered forms of antibodies include, forexample, minibodies, diabodies, diabodies fused to CH3 molecules,tetravalent antibodies, intradiabodies (e.g., Jendreyko et al. 2003. J.Biol. Chem. 278:47813), bispecific antibodies, fusion proteins (e.g.,antibody cytokine fusion proteins) or, bispecific antibodies. Otherimmunoglobulins (Ig) and certain variants thereof are described, forexample in U.S. Pat. No. 4,745,055; EP 256,654; Faulkner et al., Nature298:286 (1982); EP 120,694; EP 125,023; Morrison, J. Immun. 123:793(1979); Kohler et al., Proc. Natl. Acad. Sci. USA 77:2197 (1980); Rasoet al., Cancer Res. 41:2073 (1981); Morrison et al., Ann. Rev. Immunol.2:239 (1984); Morrison, Science 229:1202 (1985); Morrison et al., Proc.Natl. Acad. Sci. USA 81:6851 (1984); EP 255,694; EP 266,663; and WO88/03559. Reassorted immunoglobulin chains also are known. See, forexample, U.S. Pat. No. 4,444,878; WO 88/03565; and EP 68,763 andreferences cited therein.

In one embodiment, the modified antibodies of the invention areminibodies. Minibodies are dimeric molecules made up of two polypeptidechains each comprising an ScFv molecule (a single polypeptide comprisingone or more antigen binding sites, e.g., a VL domain linked by aflexible linker to a VH domain fused to a CH3 domain via a connectingpeptide.

ScFv molecules can be constructed in a VH-linker-VL orientation orVL-linker-VH orientation.

The flexible hinge that links the VL and VH domains that make up theantigen binding site preferably comprises from about 10 to about 50amino acid residues. An exemplary connecting peptide for this purpose is(Gly4Ser)3 (SEQ ID NO:17) (Huston et al. 1988. Proc. Natl. Acad. Sci.USA 85:5879). Other connecting peptides are known in the art.

Methods of making single chain antibodies are well known in the art,e.g., Ho et al. 1989. Gene 77:51; Bird et al. 1988 Science 242:423;Pantoliano et al. 1991. Biochemistry 30:10117; Milenic et al. 1991.Cancer Research 51:6363; Takkinen et al. 1991. Protein Engineering4:837.

Minibodies can be made by constructing an ScFv component and connectingpeptide-CH3 component using methods described in the art (see, e.g.,U.S. Pat. No. 5,837,821 or WO 94/09817A1). These components can beisolated from separate plasmids as restriction fragments and thenligated and recloned into an appropriate vector. Appropriate assemblycan be verified by restriction digestion and DNA sequence analysis.

Diabodies are similar to scFv molecules, but usually have a short (lessthan 10 and preferably 1-5) amino acid residue linker connecting bothV-domains, such that the VL and VH domains on the same polypeptide chaincan not interact. Instead, the VL and VH domain of one polypeptide chaininteract with the VH and VL domain (respectively) on a secondpolypeptide chain (WO 02/02781). In one embodiment, a binding moleculeof the invention is a diabody fused to at least one heavy chain portion.In a preferred embodiment, a binding molecule of the invention is adiabody fused to a CH3 domain.

Other forms of modified antibodies are also within the scope of theinstant invention (e.g., WO 02/02781 A1; U.S. Pat. Nos. 5,959,083;6,476,198 B1; US 2002/0103345 A1; WO 00/06605; Byrn et al. 1990. Nature.344:667-70; Chamow and Ashkenazi. 1996. Trends Biotechnol. 14:52).

In one embodiment, a binding molecule of the invention comprises animmunoglobulin constant region. It is known in the art that the constantregion mediates several effector functions. For example, binding of theC1 component of complement to binding molecules activates the complementsystem. Activation of complement is important in the opsonisation andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and may also be involved in autoimmunehypersensitivity. Further, binding molecules bind to cells via the Fcregion, with a Fc receptor site on the binding molecule Fc regionbinding to a Fc receptor (FcR) on a cell. There are a number of Fcreceptors which are specific for different classes of binding molecule,including IgG (gamma receptors), IgE (epsilon receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of binding molecule to Fcreceptors on cell surfaces triggers a number of important and diversebiological responses including engulfment and destruction of bindingmolecule-coated particles, clearance of immune complexes, lysis ofbinding molecule-coated target cells by killer cells (calledantibody-dependent cell-mediated cytotoxicity, or ADCC), release ofinflammatory mediators, placental transfer and control of immunoglobulinproduction.

In one embodiment, effector functions may be eliminated or reduced byusing a constant region of an IgG4 binding molecule, which is thought tobe unable to deplete target cells, or making Fc variants, whereinresidues in the Fc region critical for effector function(s) are mutatedusing techniques known in the art, for example, U.S. Pat. No. 5,585,097.For example, the deletion or inactivation (through point mutations orother means) of a constant region domain may reduce Fc receptor bindingof the circulating modified binding molecule thereby increasing tumorlocalization. In other cases it may be that constant regionmodifications consistent with the instant invention moderate complimentbinding and thus reduce the serum half life and nonspecific associationof a conjugated cytotoxin. Yet other modifications of the constantregion may be used to modify disulfide linkages or oligosaccharidemoieties that allow for enhanced localization due to increased antigenspecificity or binding molecule flexibility. More generally, thoseskilled in the art will realize that binding molecules modified asdescribed herein may exert a number of subtle effects that may or maynot be readily appreciated. However the resulting physiological profile,bioavailability and other biochemical effects of the modifications, suchas tumor localization, biodistribution and serum half-life, may easilybe measured and quantified using well know immunological techniqueswithout undue experimentation.

In one embodiment, a binding molecule of the invention can bederivatized or linked to another functional molecule (e.g., anotherpeptide or protein). Accordingly, a binding molecule of the inventioninclude derivatized and otherwise modified forms of the anti-GITRbinding molecules described herein, including immunoadhesion molecules.For example, a binding molecule of the invention can be functionallylinked (by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other molecular entities, such as anotherbinding molecule (e.g., a bispecific antibody or a diabody), adetectable agent, a cytotoxic agent, a pharmaceutical agent, and/or aprotein or peptide that can mediate association of the binding moleculewith another molecule (such as a streptavidin core region or apolyhistidine tag).

One type of derivatized binding molecule is produced by crosslinking twoor more binding molecules (of the same type or of different types, e.g.,to create bispecific antibodies). Suitable crosslinkers include thosethat are heterobifunctional, having two distinctly reactive groupsseparated by an appropriate spacer (e.g.,m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional(e.g., disuccinimidyl suberate). Such linkers are available from PierceChemical Company, Rockford, Ill.

Useful detectable agents with which a binding molecule of the inventionmay be derivatized include fluorescent compounds. Exemplary fluorescentdetectable agents include fluorescein, fluorescein isothiocyanate,rhodamine, 5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrinand the like. A binding molecule may also be derivatized with detectableenzymes, such as alkaline phosphatase, horseradish peroxidase, glucoseoxidase and the like. When a binding molecule is derivatized with adetectable enzyme, it is detected by adding additional reagents that theenzyme uses to produce a detectable reaction product. For example, whenthe detectable agent horseradish peroxidase is present, the addition ofhydrogen peroxide and diaminobenzidine leads to a colored reactionproduct, which is detectable. A binding molecule may also be derivatizedwith biotin, and detected through indirect measurement of avidin orstreptavidin binding.

IV. Expression of Binding Molecules

A binding molecule of the invention can be prepared by recombinantexpression of immunoglobulin light and heavy chain genes in a host cell.To express a binding molecule recombinantly, a host cell is transfectedwith one or more recombinant expression vectors carrying DNA fragmentsencoding the immunoglobulin light and heavy chains of the bindingmolecule such that the light and heavy chains are expressed in the hostcell and, preferably, secreted into the medium in which the host cellsare cultured, from which medium a binding molecule can be recovered.Standard recombinant DNA methodologies are used to obtain antibody heavyand light chain genes, incorporate these genes into recombinantexpression vectors, and introduce the vectors into host cells, such asthose described in Sambrook, Fritsch and Maniatis (eds), MolecularCloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,(1989), Ausubel, F. M. et al. (eds.) Current Protocols in MolecularBiology, Greene Publishing Associates, (1989) and in U.S. Pat. No.4,816,397 by Boss, et al.

To express a binding molecule of the invention, DNAs encoding partial orfull-length light and heavy chains may be inserted into expressionvectors such that the genes are operatively linked to transcriptionaland translational control sequences. In this context, the term“operatively linked” means that a binding molecule gene is ligated intoa vector such that transcriptional and translational control sequenceswithin the vector serve their intended function of regulating thetranscription and translation of the binding molecule gene. In oneembodiment, the expression vector and expression control sequences arechosen to be compatible with the expression host cell used. The bindingmolecule light chain gene and the binding molecule heavy chain gene maybe inserted into separate vector or, more typically, both genes areinserted into the same expression vector. The binding molecule genes maybe inserted into the expression vector by standard methods (e.g.,ligation of complementary restriction sites on the binding molecule genefragment and vector, or blunt end ligation if no restriction sites arepresent). Prior to insertion of the binding molecule light or heavychain sequences, the expression vector may already carry bindingmolecule constant region sequences. For example, one approach toconverting VH and VL sequences to full-length binding molecule genes isto insert them into expression vectors already encoding heavy chainconstant and light chain constant regions, respectively, such that theVH segment is operatively linked to the CH segment(s) within the vectorand the VL segment is operatively linked to the CL segment within thevector. Additionally or alternatively, the recombinant expression vectorcan encode a signal peptide that facilitates secretion of the bindingmolecule chain from a host cell. The binding molecule chain gene can becloned into the vector such that the signal peptide is linked in-frameto the amino terminus of the binding molecule chain gene. The signalpeptide can be an immunoglobulin signal peptide or a heterologous signalpeptide (i.e., a signal peptide from a non-immunoglobulin protein).

In addition to the binding molecule chain genes, the recombinantexpression vectors of the invention carry regulatory sequences thatcontrol the expression of the binding molecule chain genes in a hostcell. The term “regulatory sequence” includes promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the binding molecule chaingenes. Such regulatory sequences are described, for example, in Goeddel;Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). It will be appreciated by those skilled in theart that the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, seee.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al. and U.S. Pat. No. 4,968,615 by Schaffner, et al.

In addition to the binding molecule chain genes and regulatorysequences, the recombinant expression vectors of the invention may carryadditional sequences, such as sequences that regulate replication of thevector in host cells (e.g., origins of replication) and selectablemarker genes. The selectable marker gene facilitates selection of hostcells into which the vector has been introduced (see e.g., U.S. Pat.Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). Forexample, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin or methotrexate, on a host cell intowhich the vector has been introduced. Preferred selectable marker genesinclude the dihydrofolate reductase (DHFR) gene (for use in dhfr⁻ hostcells with methotrexate selection/amplification) and the neo gene (forG418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding the binding molecule heavy and light chains is transfected intoa host cell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection and the like. It is possible toexpress a binding molecule of the invention in either prokaryotic oreukaryotic host cells, expression of binding molecules in eukaryoticcells, and most preferably mammalian host cells, is the most preferredbecause such eukaryotic cells, and in particular mammalian cells, aremore likely than prokaryotic cells to assemble and secrete a properlyfolded and immunologically active binding molecule.

Commonly, expression vectors contain selection markers (e.g.,ampicillin-resistance, hygromycin-resistance, tetracycline resistance orneomycin resistance) to permit detection of those cells transformed withthe desired DNA sequences (see, e.g., Itakura et al., U.S. Pat. No.4,704,362).

E. coli is one prokaryotic host particularly useful for cloning thepolynucleotides (e.g., DNA sequences) of the present invention. Othermicrobial hosts suitable for use include bacilli, such as Bacillussubtilus, and other enterobacteriaceae, such as Salmonella, Serratia,and various Pseudomonas species. In these prokaryotic hosts, one canalso make expression vectors, which will typically contain expressioncontrol sequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

Other microbes, such as yeast, are also useful for expression.Saccharomyces is a preferred yeast host, with suitable vectors havingexpression control sequences (e.g., promoters), an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

In addition to microorganisms, mammalian tissue cell culture may also beused to express and produce the polypeptides of the present invention(e.g., polynucleotides encoding binding molecules). See Winnacker, FromGenes to Clones, VCH Publishers, N.Y., N.Y. (1987). Eukaryotic cells areactually preferred, because a number of suitable host cell lines capableof secreting heterologous proteins (e.g., intact binding molecules) havebeen developed in the art, and include CHO cell lines, various Cos celllines, HeLa cells, myeloma cell lines, or transformed B-cells orhybridomas. Preferably, the cells are nonhuman. Expression vectors forthese cells can include expression control sequences, such as an originof replication, a promoter, and an enhancer (Queen et al., Immunol. Rev.89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from immunoglobulin genes, SV40,adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Coet al., J. Immunol. 148:1149 (1992).

Alternatively, binding molecule-coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,Deboer et al., U.S. Pat. No. 5,741,957, Rosen, U.S. Pat. No. 5,304,489,and Meade et al., U.S. Pat. No. 5,849,992). Suitable transgenes includecoding sequences for light and/or heavy chains in operable linkage witha promoter and enhancer from a mammary gland specific gene, such ascasein or beta lactoglobulin.

Preferred mammalian host cells for expressing the recombinant bindingmolecules of the invention include Chinese Hamster Ovary (CHO cells)(including dhfr− CHO cells, described in Urlaub and Chasin, (1980) Proc.Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker,e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NS0 myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding binding molecule genes areintroduced into mammalian host cells, binding molecules are produced byculturing the host cells for a period of time sufficient to allow forexpression of the binding molecule in the host cells or, morepreferably, secretion of the binding molecule into the culture medium inwhich the host cells are grown. Binding molecules can be recovered fromthe culture medium using standard protein purification methods.

The vectors containing the polynucleotide sequences of interest (e.g.,the binding molecule heavy and light chain encoding sequences andexpression control sequences) can be transferred into the host cell bywell-known methods, which vary depending on the type of cellular host.For example, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment, electroporation,lipofection, biolistics or viral-based transfection may be used forother cellular hosts. (See generally Sambrook et al., Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989)(incorporated by reference in its entirety for all purposes). Othermethods used to transform mammalian cells include the use of polybrene,protoplast fusion, liposomes, electroporation, and microinjection (seegenerally, Sambrook et al., supra). For production of transgenicanimals, transgenes can be microinjected into fertilized oocytes, or canbe incorporated into the genome of embryonic stem cells, and the nucleiof such cells transferred into enucleated oocytes.

When heavy and light chains are cloned on separate expression vectors,the vectors are co-transfected to obtain expression and assembly ofintact immunoglobulins. Once expressed, the whole binding molecules,their dimers, individual light and heavy chains, or other immunoglobulinforms of the present invention can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns, column chromatography, HPLC purification, gelelectrophoresis and the like (see generally Scopes, Protein Purification(Springer-Verlag, N.Y., (1982)). Substantially pure binding molecules ofat least about 90 to 95% homogeneity are preferred, and 98 to 99% ormore homogeneity most preferred, for pharmaceutical uses.

Host cells can also be used to produce portions of intact bindingmolecules, such as Fab fragments or scFv molecules. It will beunderstood that variations on the above procedure are within the scopeof the present invention. For example, it may be desirable to transfecta host cell with DNA encoding either the light chain or the heavy chain(but not both) of a binding molecule of this invention. Recombinant DNAtechnology may also be used to remove some or all of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to GITR. The molecules expressed from such truncated DNAmolecules are also encompassed by a binding molecule of the invention.In addition, bifunctional binding molecules may be produced in which oneheavy and one light chain are a binding molecule of the invention andthe other heavy and light chain are specific for an antigen other thanGITR by crosslinking a binding molecule of the invention to a secondbinding molecule by standard chemical crosslinking methods.

In view of the foregoing, another aspect of the invention pertains tonucleic acid, vector and host cell compositions that can be used forrecombinant expression of a binding molecule of the invention. Thenucleotide sequence encoding the 6C8 light chain variable region isshown in FIG. 18 and SEQ ID NO.: 10. The CDR1 domain of the VLencompasses nucleotides 130-162 of SEQ ID NO:10 (SEQ ID NO:14), the CDR2domain encompasses nucleotides 208-228 of SEQ ID NO:10 (SEQ ID NO:15)and the CDR3 domain encompasses nucleotides 325-351 of SEQ ID NO:10 (SEQID NO:16). The nucleotide sequence encoding the 6C8 heavy chain variableregion is also shown in FIG. 18 and SEQ ID NO.: 9. The CDR1 domain ofthe VH encompasses nucleotides 133-168 of SEQ ID NO:9 (SEQ ID NO:11),the CDR2 domain encompasses nucleotides 211-258 of SEQ ID NO:9 (SEQ IDNO:12) and the CDR3 domain encompasses nucleotides 355-381 of SEQ IDNO:9 (SEQ ID NO:13). In one embodiment, the nucleotide sequence encodingCDR2 of the VH comprises SEQ ID NO:12. In another embodiment, thenucleotide sequence encoding CDR2 of the VH comprises SEQ ID NO:65(CACATTTGGTGGGATGATGATAAGTACTAT

CAACCATCCCTGAAGAGC). It will be appreciated by the skilled artisan thatnucleotide sequences encoding 6C8-related binding molecules can bederived from the nucleotide sequences encoding the 6C8 VL and VH usingthe genetic code and standard molecular biology techniques.

In one embodiment, the invention provides isolated nucleic acidmolecules encoding a polypeptide sequence comprising a 6C8 CDR, e.g.,comprising an amino acid sequence selected from the group consisting of:SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:19, SEQ ID NO:5, SEQ ID NO: 6, SEQID NO: 7, SEQ ID NO: 8.

In still another embodiment, the invention provides an isolated nucleicacid molecule encoding a binding molecule light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 2, although the skilledartisan will appreciate that due to the degeneracy of the genetic code,other nucleic acid molecules can encode the amino acid sequence of SEQID NO: 2. The nucleic acid molecule can encode only the VL or can alsoencode a binding molecule light chain constant region, operativelylinked to the VL. In one embodiment, this nucleic acid molecule is in arecombinant expression vector.

In still another embodiment, the invention provides an isolated nucleicacid molecule encoding a binding molecule heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 1, although the skilledartisan will appreciate that due to the degeneracy of the genetic code,other nucleic acid molecules can encode the amino acid sequence of SEQID NO: 1. In another embodiment, the invention provides an isolatednucleic acid molecule encoding a binding molecule heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 66, although theskilled artisan will appreciate that due to the degeneracy of thegenetic code, other nucleic acid molecules can encode the amino acidsequence of SEQ ID NO: 66. The nucleic acid molecule can encode only theVH or can also encode a heavy chain constant region, operatively linkedto the VH. For example, the nucleic acid molecule can comprise an IgG1or IgG2 constant region. In one embodiment, this nucleic acid moleculeis in a recombinant expression vector.

The invention also provides recombinant expression vectors encoding abinding molecule heavy chain and/or a binding molecule light chain. Forexample, in one embodiment, the invention provides a recombinantexpression vector encoding:

a) a binding molecule light chain having a variable region comprisingthe amino acid sequence of SEQ ID NO: 2; and

b) a binding molecule heavy chain having a variable region comprisingthe amino acid sequence of SEQ ID NO: 1.

In another embodiment, the invention provides a recombinant expressionvector encoding:

a) a binding molecule light chain having a variable region comprisingthe amino acid sequence of SEQ ID NO: 2; and

b) a binding molecule heavy chain having a variable region comprisingthe amino acid sequence of SEQ ID NO: 66.

The invention also provides host cells into which one or more of therecombinant expression vectors of the invention have been introduced.Preferably, the host cell is a mammalian host cell.

Still further the invention provides a method of synthesizing arecombinant binding molecules of the invention by culturing a host cellof the invention in a suitable culture medium until a recombinantbinding molecule of the invention is synthesized. The method may furthercomprise isolating the recombinant binding molecule from the culturemedium.

V. Uses of Binding Molecules of the Invention

Given their ability to bind to GITR, the binding molecules of theinvention may be used to detect GITR (e.g., in a biological sample, suchas serum or plasma), using a conventional immunoassay, such as an enzymelinked immunosorbent assays (ELISA), a radioimmunoassay (RIA) or tissueimmunohistochemistry. The invention provides a method for detectinghGITR in a biological sample comprising contacting a biological samplewith a binding molecule of the invention and detecting either thebinding molecule bound to hGITR or unbound binding molecule, to therebydetect hGITR in the biological sample. The method may be performed invitro or in vivo. The binding molecule is directly or indirectly labeledwith a detectable substance to facilitate detection of the bound orunbound binding molecule. Suitable detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescent materialsand radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; and examples of suitable radioactive material include ¹²⁵I,¹³¹I, ³⁵S or ³H.

Alternative to labeling the binding molecule, hGITR can be assayed inbiological fluids by a competition immunoassay utilizing GITR standardslabeled with a detectable substance and an unlabeled anti-hGITR bindingmolecule. In this assay, the biological sample, the labeled GITRstandards and the anti-hGITR binding molecule are combined and theamount of labeled GITR standard bound to the unlabeled binding moleculeis determined. The amount of hGITR in the biological sample is inverselyproportional to the amount of labeled GITR standard bound to theanti-hGITR binding molecule.

An anti-GITR binding molecule of the invention can also be used todetect GITRs in samples from species other than humans, in particularGITRs from primates (e.g., chimpanzee, baboon, marmoset, cynomolgus andrhesus).

In another embodiment, the invention provides a method for abrogatingthe suppression of T effector cells by T regulatory cells. Abrogation ofsuppression of T effector cells by T regulatory cells can be assayed,for example, by measuring the ability of the binding molecule to enhanceT cell effector function in the presence of T regulatory cells, e.g.,cytokine production, (e.g., IL-2 production) or cell proliferation(e.g., T helper cell proliferation), by, for example, measuring³H-thymidine incorporation or by FACS analysis. For example, theresponse or activity of T effector cells will be low in the presence ofT regulatory cells, but will increase with the addition of a GITRbinding molecule even if T regulatory cells are present, i.e., a GITRbinding molecules abrogates the suppression of T effector cells by Tregulatory cells.

The binding molecules of the invention may also be used to attenuate thedegradation of I-κB in cells. Attenuated degradation of I-κB in cellscan be assayed, for example, by Western blotting and quantitating theamount of I-κB following treatment of cells with anti-GITR bindingmolecule.

Numerous disease or pathological conditions would benefit from enhancingthe activity of T effector cells and/or downmodulating the activity of Tregulatory cells, e.g., by abrogating the suppression of T effectorcells by T regulatory cells. For example, immune effector cells oftenfail to react effectively with cancer cells. Accordingly, when anenhanced effector T cell or antibody response is desired, the methods ofthe invention can be used to treat a subject suffering from such adisorder. In one embodiment such methods comprise administering to thesubject a binding molecule of the invention such that suppression of Teffector cells by T regulatory cells is abrogated, thereby enhancing animmune response. Preferably, the subject is a human subject.Alternatively, the subject can be a mammal expressing a GITR with whicha binding molecule of the invention cross-reacts. Still further, thesubject can be a mammal into which GITR has been introduced (e.g., byadministration of GITR or by expression of a GITR transgene). A bindingmolecule of the invention may be administered to a human subject fortherapeutic or prophylactic purposes. For example, the subject may havebeen diagnosed as having the disease or disorder or may be predisposedor susceptible to the disease. Moreover, a binding molecule of theinvention can be administered to a non-human mammal expressing a GITRmolecule with which the binding molecule cross-reacts (e.g., a primate)for veterinary purposes or as an animal model of human disease.Regarding the latter, such animal models may be useful for evaluatingthe therapeutic and/or prophylactic efficacy of binding molecules of theinvention (e.g., testing of dosages and/or time courses ofadministration).

Exemplary uses of the binding molecules of the invention are discussedfurther below:

Immunostimulatory Compositions

As described in the appended examples, the binding molecules of theinvention can be used as immunostimulatory compositions (or vaccines),e.g., in combination with an antigen, to promote an enhanced immuneresponse to an antigen of interest, e.g., a protein antigen, in asubject. That is, the binding molecules of the invention can serve asadjuvants to enhance immune responses. For example, to stimulate anantibody or cellular immune response to an antigen of interest (e.g.,for vaccination purposes), the antigen and a binding molecules of theinvention can be coadministered (e.g., coadministered at the same timein the same or separate compositions, or sequentially in time) such thatan enhanced immune response occurs. The antigen of interest and abinding molecule can be formulated together into a single pharmaceuticalcomposition or in separate compositions. In one embodiment, the antigenof interest and the binding molecule are administered simultaneously tothe subject. Alternatively, in certain situations it may be desirable toadminister the antigen first and then the binding molecule or vice versa(for example, it may be beneficial to first administer the antigen aloneto stimulate a response and then administer a binding molecule, alone ortogether with a boost of antigen). In preferred embodiments, a GITRbinding molecule of the invention is administered at the time of primingwith antigen, i.e., at the time of the first administration of antigen.For example, day −3, −2, −1, 0, +1, +2, +3. A particularly preferred dayof administration of a GITR binding molecule of the invention is day −1prior to administration of antigen.

An antigen of interest is, for example, one capable of providingprotection in subject against challenge by an infectious agent fromwhich the antigen was derived, or which is capable of affecting tumorgrowth and metastasis in a manner which is of benefit to a subject.Exemplary antigens of interest therefore include those derived frominfectious agents, cancer cells, and the like, wherein an immuneresponse directed against the antigen serves to prevent or treat diseasecaused by the agent. Such antigens include, but are not limited to,viral, bacterial, fungal or parasite proteins, glycoproteins,lipoproteins, glycolipids, and the like. Antigens of interest alsoinclude those which provide benefit to a subject which is at risk foracquiring or which is diagnosed as having a tumor and may include, e.g.,tumor-related antigens expressed exclusively by or at increased levelsby tumor cells. The subject is preferably a mammal and most preferably,is a human.

As used herein the term “pathogen” or “pathogenic agent” includesmicroorganisms that are capable of infecting or parasitizing normalhosts (e.g., animals (such as mammals, preferably primates, e.g.humans)). As used herein, the term also includes opportunistic agents,e.g., microorganisms that are capable of infecting or parasitizingabnormal hosts, e.g., hosts in which normal flora have been supplanted,e.g., as a result of a treatment regimen, or immunocompromised hosts. Asused herein the term also includes microorganisms whose replication isunwanted in a subject or toxic molecules (e.g., toxins) produced bymicroorganisms.

Non-limiting examples of viral antigens include, but are not limited to,the nucleoprotein (NP) of influenza virus and the Gag proteins of HIV.Other heterologous antigens include, but are not limited to, HIV Envprotein or its component parts, gp120 and gp41, HIV Nef protein, and theHIV Pol proteins, reverse transcriptase and protease. In addition, otherviral antigens such as Ebola virus (EBOV) antigens, such as, forexample, EBOV NP or glycoprotein (GP), either full-length or GP deletedin the mucin region of the molecule (Yang Z-Y, et al. (2000) Nat Med6:886-9, 2000), small pox antigens, hepatitis A, B or C virus, humanrhinovirus such as type 2 or type 14, Herpes simplex virus, poliovirustype 2 or 3, foot-and-mouth disease virus (FMDV), rabies virus,rotavirus, influenza virus, coxsackie virus, human papilloma virus(HPV), for example the type 16 papilloma virus, the E7 protein thereof,and fragments containing the E7 protein or its epitopes; and simianimmunodeficiency virus (SIV) may be used. The antigens of interest neednot be limited to antigens of viral origin. Parasitic antigens, such as,for example, malarial antigens are included, as are fungal antigens,bacterial antigens and tumor antigens can also be used in connectionwith the disclosed compositions and methods. Non-limiting examples ofbacterial antigens include: Bordetella pertussis (e.g., P69 protein andfilamentous haemagglutinin (FHA) antigens), Vibrio cholerae, Bacillusanthracis, and E. coli antigens such as E. coli heat Labile toxin Bsubunit (LT-B), E. coli K88 antigens, and enterotoxigenic E. coliantigens. Other examples of antigens include Schistosoma mansoni P28glutathione S-transferase antigens (P28 antigens) and antigens offlukes, mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, andmalaria parasites, e.g., parasites of the genus plasmodium or babesia,for example Plasmodium falciparum, and peptides encoding immunogenicepitopes from the aforementioned antigens.

An infection, disease or disorder which may be treated or prevented bythe administration of a vaccine of the invention includes any infection,disease or disorder wherein a host immune response acts to prevent theinfection, disease or disorder. Diseases, disorders, or infection whichmay be treated or prevented by the administration of theimmunostimulatory compositions of the invention include, but are notlimited to, any infection, disease or disorder caused by or related to afungus, parasite, virus, or bacteria, diseases, disorders or infectionscaused by or related to various agents used in bioterrorism,listeriosis, Ebola virus, SARS, small pox, hepatitis A, hepatitis B,hepatitis C, and hepatitis E, diseases and disorders caused by humanrhinovirus, HIV (e.g., HIV-1 and HIV-2), and AIDS, Herpes, polio,foot-and-mouth disease, rabies, diseases or disorders caused by orrelated to: rotavirus, influenza, coxsackie virus, human papillomavirus, SIV, malaria, cancer, e.g., tumors, human herpes viruses,cytomegalovirus (esp. Human), Epstein-Barr virus, Varicella ZosterVirus, hepatitis viruses, such as hepatitis B virus, hepatitis A virus,hepatitis C virus a, paramyxoviruses: Respiratory Syncytial virus,parainfluenza virus, measles virus, mumps virus, human papilloma viruses(for example HPV6, 11, 16, 18, and the like), flaviviruses (e.g. YellowFever Virus, Dengue Virus, Tick-borne encephalitis virus, JapaneseEncephalitis Virus), or influenza virus, e.g., influenza A (e.g.,subtypes, hemagglutinin (H) and neuraminidase (N)), influenza B, andinfluenza C, and diseases or disorders caused by or related to infectionby bacterial organisms, including gram-positive and gram-negativebacteria. Examples include, but are not limited to, Neisseria spp,including N. gonorrhea and N. meningitidis, Streptococcus spp, includingS. pneumoniae, S. pyogenes, S. agalactiae, S. mutans; Haemophilus spp,including H. influenzae type B, non typeable H. influenzae, H. ducreyi;Moraxella spp, including M catarrhalis, also known as Branhamellacatarrhalis; Bordetella spp, including B. pertussis, B. parapertussisand B. bronchiseptica; Mycobacterium spp., including M. tuberculosis, M.bovis, M. leprae, M. avium, M. paratuberculosis, M. smegmatis;Legionella spp, including L. pneumophila; Escherichia spp, includingenterotoxic E. coli, enterohemorragic E. coli, enteropathogenic E. coli;Vibrio spp, including V. cholera, Shigella spp, including S. sonnei, S.dysenteriae, S. flexnerii; Yersinia spp, including Y. enterocolitica, Y.pestis, Y. pseudotuberculosis, Campylobacter spp, including C. jejuniand C. coli; Salmonella spp, including S. typhi, S. paratyphi, S.choleraesuis, S. enteritidis; Listeria spp., including L. monocytogenes;Helicobacter spp, including H. pylori; Pseudomonas spp, including P.aeruginosa, Staphylococcus spp., including S. aureus, S. epidermidis;Enterococcus spp., including E. faecalis, E. faecium; Clostridium spp.,including C. tetani, C. botulinum, C. difficile; Bacillus spp.,including B. anthracis; Corynebacterium spp., including C. diphtherias;Borrelia spp., including B. burgdorferi, B. garinii, B. afzelii, B.andersonii, B. hermsii; Ehrlichia spp., including E. equi and the agentof the Human Granulocytic Ehrlichiosis; Rickettsia spp, including R.rickettsii; Chlamydia spp., including C. trachomatis, C. neumoniae, C.psittaci; Leptospira spp., including L. interrogans; Treponema spp.,including T. pallidum, T. denticola, T. hyodysenteriae. Preferredbacteria include, but are not limited to, Listeria, mycobacteria,mycobacteria (e.g., tuberculosis), Anthrax, Salmonella and Listeriamonocytogenes, Bordetella pertussis, Vibrio cholerae, flukes,mycoplasma, roundworms, tapeworms, Chlamydia trachomatis, and malariaparasites,.

As used herein, the term “bacterial infections” include infections witha variety of In another embodiment, T cells can be removed from apatient, and contacted in vitro with an anti-GITR binding molecule,optionally with an activating signal (e.g., antigen plus APCs or apolyclonal antibody) and reintroduced into the patient.

Regulatory T cells play an important role in the maintenance ofimmunological self-tolerance by suppressing immune responses againstautoimmune diseases and cancer. Accordingly, in one embodiment,abrogating the suppression of T effector cells by T regulatory cellswould be beneficial for enhancing an immune response in cancer.Therefore, the binding molecules of the invention can be used in thetreatment of malignancies, to inhibit tumor growth or metastasis. Thebinding molecules may be administered systemically or locally to thetumor site.

In one embodiment, modulation of GITR function may be useful in theinduction of tumor immunity, i.e., for the treatment of a subject with aneoplastic disease or cancer. In one embodiment, a binding molecule ofthe invention reduces tumor size, inhibits tumor growth and/or prolongsthe survival time of a tumor-bearing subject. A GITR binding moleculecan be administered to a patient having tumor cells (e.g., sarcoma,melanoma, lymphoma, leukemia, neuroblastoma, carcinoma) to overcometumor-specific tolerance in the subject.

By the term “tumor-related antigen,” as used herein, is meant an antigenwhich affects tumor growth or metastasis in a host organism. Thetumor-related antigen may be an antigen expressed by a tumor cell, or itmay be an antigen which is expressed by a non-tumor cell, but which whenso expressed, promotes the growth or metastasis of tumor cells. Thetypes of tumor antigens and tumor-related antigens include any known orheretofore unknown tumor antigen, including, without limitation, thebcr/abl antigen in leukemia, HPVE6 and E7 antigens of the oncogenicvirus associated with cervical cancer, the MAGE1 and MZ2-E antigens inor associated with melanoma, and the MVC-1 and HER-2 antigens in orassociated with breast cancer.

As used herein, the term “neoplastic disease” is characterized bymalignant tumor growth or in disease states characterized by benignhyperproliferative and hyperplastic cells. The common medical meaning ofthe term “neoplasia” refers to “new cell growth” that results as a lossof responsiveness to normal growth controls, e.g., neoplastic cellgrowth.

As used herein, the terms “hyperproliferative”, “hyperplastic”,malignant” and “neoplastic” are used interchangeably, and refer to thosecells in an abnormal state or condition characterized by rapidproliferation or neoplasia. The terms are meant to include all types ofhyperproliferative growth, hyperplastic growth, cancerous growths oroncogenic processes, metastatic tissues or malignantly transformedcells, tissues, or organs, irrespective of histopathologic type or stageof invasiveness. A “hyperplasia” refers to cells undergoing anabnormally high rate of growth. However, as used herein, the termsneoplasia and hyperplasia can be used interchangeably, as their contextwill reveal, referring generally to cells experiencing abnormal cellgrowth rates. Neoplasias and hyperplasias include “tumors,” which may beeither benign, premalignant or malignant.

The terms “neoplasia,” “hyperplasia,” and “tumor” are often commonlyreferred to as “cancer,” which is a general name for more than 100disease that are characterized by uncontrolled, abnormal growth ofcells. Examples of cancer include, but are not limited to: breast;colon; non-small cell lung, head and neck; colorectal; lung; prostate;ovary; renal; melanoma; and gastrointestinal (e.g., pancreatic andstomach) cancer; and osteogenic sarcoma.

In one embodiment, the cancer is selected from the group consisting of:pancreatic cancer, melanomas, breast cancer, lung cancer, bronchialcancer, colorectal cancer, prostate cancer, stomach cancer, ovariancancer, urinary bladder cancer, brain or central nervous system cancer,peripheral nervous system cancer, esophageal cancer, cervical cancer,uterine or endometrial cancer, cancer of the oral cavity or pharynx,liver cancer, kidney cancer, testicular cancer, biliary tract cancer,small bowel or appendix cancer, salivary gland cancer, thyroid glandcancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, cancer ofhematological tissues.

Accordingly, this invention also relates to a method of treatingneoplastic disease or cancer in a subject, preferably a human, or otheranimal by administering to such subject or animal an effective amount ofa binding molecule of the invention. One skilled in the art is able, byroutine experimentation, to determine what an effective amount ofpolypeptide would be for the purpose of treating neoplastic disease orcancer. For example, a therapeutically effective amount of a bindingmolecule of the invention may vary according to factors such as thedisease stage (e.g., stage I versus stage IV), age, sex, medicalcomplications (e.g., immunosuppressed conditions or diseases) and weightof the subject, and the ability of the binding molecule to elicit adesired response in the subject. The dosage regimen may be adjusted toprovide the optimum therapeutic and/or prophylactic response. Forexample, several divided doses may be administered daily, or the dosemay be proportionally reduced as indicated by the exigencies of thetherapeutic situation. Generally, however, an effective dosage isexpected to be in the range of about 0.05 to 100 milligrams per kilogrambody weight per day and more preferably from about 0.5 to 10, milligramsper kilogram body weight per day.

Methods of Enhancing Immune Responses

The subject binding molecules may also be used in methods of enhancingimmune responses. Upregulation of immune responses may be in the form ofenhancing an existing immune response or eliciting an initial immuneresponse. For example, enhancing an immune response by modulation ofGITR may be useful in cases of viral infection. As anti-GITR bindingmolecules act to enhance immune responses, they would be therapeuticallyuseful in situations where more rapid or thorough clearance ofpathogenic agents, e.g., bacteria and viruses would be beneficial.Accordingly, the anti-GITR binding molecules of the invention may beused therapeutically, either or alone or in combination with an antigenor an additional immunostimulatory agent, to treat a subject sufferingfrom a disease or disorder, such as an infectious disease or malignancy,e.g., those listed supra.

Anti-GITR binding molecules may also be used prophylactically invaccines against various pathogens. Immunity against a pathogen, e.g., avirus, could be induced by vaccinating with a viral protein along with aGITR binding molecule (as described above). Alternately, an expressionvector which encodes genes for both a pathogenic antigen and a GITRbinding molecule, e.g., a vaccinia virus expression vector engineered toexpress a nucleic acid encoding a viral protein and a nucleic acidencoding a GITR binding molecule, can be used for vaccination. Pathogensfor which vaccines may be useful include, for example, hepatitis B,hepatitis C, Epstein-Barr virus, cytomegalovirus, HIV-1, HIV-2,influenza, tuberculosis, malaria and schistosomiasis.

The present invention is further directed to binding molecule-basedtherapies which involve administering binding molecules of the inventionto an animal, preferably a mammal, and most preferably a human, patientfor treating, detecting, and/or preventing one or more of the discloseddiseases, disorders, or conditions. Therapeutic compounds of theinvention include, but are not limited to, binding molecules of theinvention (including analogs and derivatives thereof as describedherein) and anti-idiotypic binding molecules as described herein. Abinding molecule of the invention can be used to treat, diagnose,inhibit or prevent diseases, disorders or conditions associated withaberrant activity of GITR, including, but not limited to, any one ormore of the diseases, disorders, or conditions described herein (e.g.,binding molecules of the invention may be provided in pharmaceuticallyacceptable compositions as known in the art or as described herein.

A binding molecule of this invention may be advantageously utilized incombination with other monoclonal or chimeric binding molecules, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with a binding molecule.

A binding molecule of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents,antibiotics, therapy directed against a pathogenic agent (such as forexample an immunotherapeutic or chemotherapeutic agent effective againsta viral pathogen or a bacterial antigen) and immunostimulatory agents. Abinding molecule of the invention may also be administered incombination with an antigen to which an enhanced immune response isdesired, e.g., a vaccine or an antigen from a pathogenic agent (or anattenuated form of a virus or bacterium) or an antigen from a tumor asdescribed above. In one embodiment, a binding molecule of the inventionis administered alone or in a combination therapy to a subject with aninfection. In another embodiment, a binding molecule of the invention isadministered alone or in combination to a subject with a chronic viralinfection. In yet another embodiment, a binding molecule of theinvention are administered alone or in combination to a subject withcancer.

Generally, administration of binding molecules derived a species that isthe same species as that of the patient is preferred. Thus, in apreferred embodiment, human binding molecules, derivatives, analogs, ornucleic acids, are administered to a human patient for therapy orprophylaxis.

VI. Pharmaceutical Compositions

A binding molecule of the invention can be incorporated intopharmaceutical compositions suitable for administration to a subject.Typically, the pharmaceutical composition comprises a binding moleculeof the invention and a pharmaceutically acceptable carrier. As usedherein the language “pharmaceutically acceptable carrier” includessolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacturer and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is preferable to include isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, and sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, a binding molecule of the invention is prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations should be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensionscan also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

VII. Administration of Binding Molecules of the Invention

Binding molecules of the invention are contacted with cells from asubject in a biologically compatible form in vitro or in vivo. By“biologically compatible form” is meant a form of the agent to beadministered in which any toxic effects are outweighed by thetherapeutic effects of the binding molecule.

In one embodiment, the subject compositions are administered to asubject. Administration of a therapeutically active amount of thetherapeutic compositions of the present invention is defined as anamount effective, at dosages and for periods of time necessary toachieve the desired result. For example, a therapeutically active amountof binding molecule may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of thebinding molecule to elicit a desired response in the individual. Dosageregimens can be adjusted to provide the optimum therapeutic response.For example, several divided doses can be administered daily or the dosecan be proportionally reduced as indicated by the exigencies of thetherapeutic situation.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of a binding molecule of the invention. A “therapeuticallyeffective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the binding molecule may varyaccording to factors such as the disease state, age, sex, and weight ofthe individual, and the ability of the binding molecule to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of the bindingmolecule are outweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic or prophylactic response). For example, a singlebolus may be administered, several divided doses may be administeredover time or the dose may be proportionally reduced or increased asindicated by the exigencies of the therapeutic situation. It isespecially advantageous to formulate parenteral compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a binding molecule of the inventionis, e.g., from about 0.1-25 mg/kg, from about 1.0-10 mg/kg, from about0.5-2.5 mg/kg, from about 5-25 mg/kg, from about 1-400 mg/kg. It is tobe noted that dosage values may vary with the type and severity of thecondition to be alleviated. It is to be further understood that for anyparticular subject, specific dosage regimens should be adjusted overtime according to the individual need and the professional judgment ofthe person administering or supervising the administration of thecompositions, and that dosage ranges set forth herein are exemplary onlyand are not intended to limit the scope or practice of the claimedcomposition. Additional, non-limiting ranges for a therapeutically orprophylactically effective amount of a binding molecule of the inventionis from about 0.0001 to 100 mg/kg, and from about 0.01 to 5 mg/kg (e.g.,0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2 mg/kg, etc.),of the subject body weight. For example, dosages can be 1 mg/kg bodyweight or 10 mg/kg body weight or within the range of 1-10 mg/kg,preferably at least 1 mg/kg. Doses intermediate in the above ranges arealso intended to be within the scope of the invention.

Subjects can be administered such doses daily, on alternative days,weekly or according to any other schedule determined by empiricalanalysis. An exemplary treatment entails administration in multipledosages over a prolonged period, for example, of at least six months.Additional exemplary treatment regimes entail administration once perevery two weeks or once a month or once every 3 to 6 months. Exemplarydosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30mg/kg on alternate days or 60 mg/kg weekly.

Binding molecules of the invention can be administered on multipleoccasions. Intervals between single dosages can be, e.g., daily, weekly,monthly or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of binding molecule in the patient. Bindingmolecules of the invention can optionally be administered in combinationwith other agents that are effective in treating the disorder orcondition in need of treatment (e.g., prophylactic or therapeutic).Preferred additional agents are those which are art recognized and arestandardly administered for a particular disorder.

The binding molecule can be administered in a convenient manner such asby injection (subcutaneous, intravenous, etc.), oral administration,inhalation, transdermal application, or rectal administration. Dependingon the route of administration, the active compound can be coated in amaterial to protect the compound from the action of enzymes, acids andother natural conditions which may inactivate the compound. For example,to administer the agent by other than parenteral administration, it maybe desirable to coat, or co-administer the agent with, a material toprevent its inactivation.

A binding molecule of the present invention can be administered by avariety of methods known in the art, although for many therapeuticapplications, the preferred route/mode of administration is intravenousinjection or infusion. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. In certain embodiments, the active compound may beprepared with a carrier that will protect the compound against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, a binding molecule of the invention may beorally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

Binding molecules can be co-administered with enzyme inhibitors or in anappropriate carrier such as liposomes. Pharmaceutically acceptablediluents include saline and aqueous buffer solutions. Adjuvant is usedin its broadest sense and includes any immune stimulating compound suchas interferon. Adjuvants contemplated herein include resorcinols,non-ionic surfactants such as polyoxyethylene oleyl ether andn-hexadecyl polyethylene ether. Enzyme inhibitors include pancreatictrypsin inhibitor, diisopropylfluorophosphate (DEEP) and trasylol.Liposomes include water-in-oil-in-water emulsions as well asconventional liposomes (Sterna et al. (1984) J Neuroimmunol. 7:27).

The active compound may also be administered parenterally orintraperitoneally. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations may contain apreservative to prevent the growth of microorganisms.

When the active compound is suitably protected, as described above, thebinding molecule can be orally administered, for example, with an inertdiluent or an assimilable edible carrier.

Supplementary active compounds can also be incorporated into thecompositions. In certain embodiments, a binding molecule of theinvention is coformulated with and/or coadministered with one or moreadditional therapeutic agents. For example, an anti-GITR bindingmolecule of the invention may be coformulated and/or coadministered withone or more additional antibodies that bind other targets e.g.,antibodies that bind other cytokines or that bind cell surfacemolecules. Such combination therapies may advantageously utilize lowerdosages of the administered therapeutic agents, thus avoiding possibletoxicities or complications associated with the various monotherapies.

The present invention further encompasses binding molecules conjugatedto a diagnostic or therapeutic agent. A binding molecule can be useddiagnostically to, for example, monitor the development or progressionof a tumor as part of a clinical testing procedure to, e.g., determinethe efficacy of a given treatment regimen. Detection can be facilitatedby coupling the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to thebinding molecule or indirectly, through an intermediate (such as, forexample, a linker known in the art) using techniques known in the art.See, for example, U.S. Pat. No. 4,741,900 for metal ions which can beconjugated to binding molecules for use as diagnostics according to thepresent invention. Examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialI¹²⁵I¹³¹, I¹¹¹, In^(99 Tc).

Further, a binding molecule may be conjugated to a therapeutic moietysuch as a cytotoxin, e.g., a cytostatic or cytocidal agent, atherapeutic agent, a radioactive metal ion, e.g., alpha-emitters suchas, for example, ²¹³Bi, biological toxins, prodrugs, peptides, proteins,enzymes, viruses, lipids, biological response modifiers, pharmaceuticalagents, immunologically active ligands (e.g., lymphokines or otherantibodies). In another embodiment, a binding molecule of the inventioncan be conjugated to a molecule that decreases vascularization oftumors. In other embodiments, the disclosed compositions may comprisebinding molecules of the invention coupled to drugs or prodrugs. Stillother embodiments of the present invention comprise the use of bindingmolecules of the invention conjugated to specific biotoxins or theircytotoxic fragments such as ricin, gelonin, pseudomonas exotoxin ordiphtheria toxin. The selection of which conjugated or unconjugatedbinding molecule to use will depend on the type and stage of cancer, useof adjunct treatment (e.g., chemotherapy or external radiation) andpatient condition. It will be appreciated that one skilled in the artcould readily make such a selection in view of the teachings herein.

A cytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carnustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

This invention is further illustrated by the following examples, whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures, are incorporated herein byreference.

EXAMPLES

The following materials and methods were used in certain Examples:

Methods

Culture of T Cell Lines

Differentiated cell lines were produced from cells prepared from humancord blood or peripheral blood CD4+CD45RA+ naïve T cells by a variety ofmethods, including flow cytometry and magnetic bead separations. Purityof the starting populations was >95%. Cells were then stimulated by CD3and CD28 antibodies in RPMI 1640 with 10% FCS and 1% Human AB serum withdefined mixtures of cytokines and neutralizing antibodies to cytokinesto produce the differentiated cell types. Th1 cells were produced byculture with IL12 (62 U/ml) and anti-IL4 (0.2 μg/ml); Th2 cells wereproduced by culture in IL4 (145 U/ml) and anti-IL12 (10 μg/ml) andanti-IFNγ (10 μg/ml); and regulatory T cells were produced by culture inTGFβ (32 U/ml), IL9 (42 U/ml), anti-IL4 (10 ug/ml) and anti-IL12 (10ug/ml) and anti-IFNγ (10 ug/ml). (Note: anti-IL12 was not used in allexperiments). All cultures were supplemented with IL2 (65 U/ml) and IL15(4500 U/ml). Cells were split into larger culture dishes as warranted bycell division.

Example 1 Isolation and Purification of 6C8

The 6C8 antibody is an IgG2b, kappa. Purification of this antibodyrevealed the presence of a double heavy chain (FIG. 1). This could havebeen due to alternative glycosylation or contamination with another Ab.Size exclusion chromatography showed the presence of one peak (FIG. 2).

The 6C8 antibody was purified as follows:

-   -   1. Washed 20 ml Protein G (Pharmacia HR 10/30) with 5CV of dPBS    -   2. Loaded 1 L (run 1) or 2 L (run 2) of hGITR (6C8) supernatant    -   3. Washed with 10 CV of dPBS    -   4. Eluted with 100 mM Citrate, pH 2.8 directly into 1 M Tris        (20-25% v: v)    -   5. Stripped with 100 mM Citrate, pH 2.8, 0.3 M NaCl

Example 2 Characterization of 6C8

The 6C8 antibody binds to GITR-L-M transfected cells (FIG. 3) andactivated PBLs (FIG. 4). The saturation curve of biotin-labeledanti-GITR on activated lymphocytes suggests a good relative affinity(FIG. 5).

The 6C8 antibody is co-stimulatory on T lymphocytes activated withsuboptimal anti-CD3 (FIG. 6). This antibody does not co-stimulate to thesame level as CD28, but it is comparable to the commercial anti-GITR(R&D).

The 6C8 antibody does not induce apoptosis on activated lymphocytes(FIG. 7). Lymphocytes were activated with PHA for 3 days prior to theaddition of the antibody. Compared to YTH 655 (anti-human CD2 known toinduce apoptosis on activated lymphocytes) 6C8 does not increase theapoptosis of activated T lymphocytes.

The 6C8 antibody does not block a primary mixed lymphocyte reaction(MLR) (FIG. 8). TRX1 (anti-human CD4) was used as a positive control forthe MLR.

Example 3 The 6C8 Antibody Abrogates Suppression of T Effector CellsInduced by T Regulatory Cells

The 6C8 antibody was able to block the suppression induced by Tregulatory cells (FIG. 9). CD4+/CD25+ cells were added to CD4+/CD25−cells at various ratios. The cells were stimulated with plate-boundanti-CD3 and anti-CD28. At a ratio of 1:1 the CD4+/CD25+ cells were ableto abrogate the proliferation of the CD4+/CD25− cells. The addition of6C8 to the cultures was able to block the suppression in adose-dependent manner.

When T cells were stimulated through anti-CD3 only (with noco-stimulation with anti-CD28) there was no suppression observed withthe addition of CD4+/CD25+ cells to the CD4+/CD25− cells, in fact, theanti-GITR antibody was slightly co-stimulatory under these conditions(FIG. 10).

Example 4 The 6C8 Antibody Modulates Signaling via NF-kB

Activation of T cells via CD3 or CD3 and CD28 results in activation ofI-κB signaling pathways, as assessed by both I-κB phosphorylation (FIGS.12 and 14) and subsequent degradation (FIGS. 11 and 13).

As presented on FIG. 11, under conditions of partial activation,anti-GITR has a significant effect on I-κB signaling, as assessed bytime dependent degradation of I-kB. In the presence of the GITR bindingmolecule, degradation is significantly attenuated, at all time pointsanalyzed. Above changes nicely correlate with decline of phosphorylationof I-κB (FIG. 12).

Interestingly the magnitude of response is greater for TH2 and Treg vs.TH1. Furthermore the expression of GITR appears to be higher on TH1cells, compared to TH2 and Treg cells, (as assessed by MCF (mean channelfluorescence)) in parallel experiments. T cells fully activated viacrosslinking to CD3 and CD28 loose their responsiveness to anti-GITR,however fully retain activation of I-κb via TNF-α.

Example 5 The 6C8 Antibody Enhances Immune Responses

The B16 melanoma tumor model is an aggressive melanoma model that hasbeen used to study the role of T regulatory cells in cancer. Treatmentof mice with a depleting anti-CD25 antibody or anti-CTLA-4 has shownpromising results in this model. In both cases, treatments were able todelay tumor on-set and tumor size. Since GITR is expressed on CD25+cells and may be involved in abrogating the suppression of T regulatorycells, B16-tumor bearing mice were treated with anti-GITR bindingmolecule to determine if there was an effect on tumor on-set or tumorsize. Treatment with anti-GITR binding molecule one day after the micewere injected with tumor resulted in a delayed onset and size of tumor(FIG. 17). In addition, there were still mice in the GITR treated groupthat were tumor-free at the end of the study.

All animals were injected with 10⁴ B16 melanoma cells in their rightflank on day 0. The GITR groups received 2 milligrams, 1 milligram, 0.5,milligrams, or 0.2 milligrams of anti-GITR binding molecule on Day 1.Measurable tumors were visible starting on Day 16.

Example 6 Simultaneous Delivery of Anti-GITR and Antigen Results in anAdjuvant Effect

The adjuvant effect of an anti-mGITR antibody on the humoral response toovalbumin (Ova) or hemagglutinin (HA) was further investigated. Micewere treated with either no antibody, YAML (isotype control), or 2F8(rat-anti-mGITR) on days −1, 0, and 1 at 0.4 mg/day. To assess theimportance of Fc receptor engagement in the mechanism of action of thebinding molecule, an additional group of animals was treated with 6mg/day of 2F8 F(ab′)2 on days −1, 0 and 1. This dose was selected basedon the short half life of F(ab′)2 compared to whole antibody. Mice wereimmunized with Ova (100 μg) or HA (10 μg) on day 0. The Ova treated micewere challenged with 100 μg Ova on day 14 and then bled on days 21 and28 to obtain serum samples for ELISA assays. HA treated mice werechallenged with 5 μg HA on day 14 and also bled on days 21 and 28.

Serum concentrations of 2F8 and 2F8 F(ab′)2 were monitored to assess thepharmacokinetic profiles of the binding molecules. On day 1, serumlevels of binding molecule in mice treated with 2F8 or the 2F8 F(ab′)2fragments were comparable. Binding molecule was detected in the 2F8treated mice until day 9, whereas the 2F8 F(ab′)2 fragment treated micehad detectable binding molecule only until day 3, despite a 15× higherdose.

The results demonstrate that in the HA arm of the study, mice treatedwith 2F8 had a 4 and 5 fold increase in anti-HA antibodies compared toanimals treated with no antibody and an 18 and 20 fold increase inanti-HA antibodies compared to YAML treated mice on days 21 and 28,respectively (FIG. 19). The anti-HA titer observed with the anti-mGITRantibody as an adjuvant is comparable to the titer observed when HA wasadministered with Incomplete Freund's adjuvant (IFA). This suggests thatthe response observed with the anti-mGITR antibody is comparable to oneof the most potent adjuvants frequently utilized in immunologicalstudies.

In the Ova arm of the study, mice treated with 2F8 had a 13 and 6 foldincrease in anti-Ova antibodies compared to animals treated with noantibody and a 17 and 8 fold increase in anti-Ova antibodies compared toYAML treated mice on day 21 and day 28, respectively (FIG. 20). Theeffect of the 2F8 antibody on the response to Ova was comparable to theobserved response to HA. Mice treated with 2F8 F(ab′)2 had a 4 and 3fold increase in Anti-Ova antibodies compared to animals treated with noantibody and a 6 and 5 fold increase in anti-Ova antibodies compared toYAML treated mice on day 21 and day 28, respectively (FIG. 20). The doseof F(ab′)2 and the different pharmacokinetic profile compared to wholeantibody may explain the decreased anti-Ova response when compared tothe 2F8 treated mice.

Together, these data demonstrate that the effect of the 2F8 antibody onthe humoral response to antigen is predominantly attributable to theF(ab′)2 portion of the antibody and that Fc receptor engagement may notbe required for the adjuvant effect of the anti-mGITR antibody.

Example 7 Preparation of a Chimeric Anti-GITR Binding Molecule

The 6C8 variable light chain region was grafted to a human light chainconstant region using conventional molecular biological techniques. TheIgG1 light chain constant region was used. The amino acid sequence ofthe complete chimeric light chain GITR binding molecule is shown below:

(SEQ ID NO: 22) DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.

The 6C8 variable heavy chain was also grafted to a human heavy chainconstant region using conventional molecular biological techniques. TheIgG1 heavy chain constant region was used. The amino acid sequence ofthe complete chimeric heavy chain GITR binding molecule is shown below(also referred to as “Gly”):

(SEQ ID NO: 23) QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Since the amino acid sequence NX(S/T) is a putative consensus sequencefor a glycosylation site which may affect the production of the bindingmolecule, and IgG1 constant region of the 6C8 heavy chain has thesequence NST, a second version of the heavy chain constant region wasprepared to conservatively substitute a glutamine for an asparagine atamino acid residue 299 (bolded and underlined above) of SEQ ID NO:23.Accordingly, a second human constant region was grafted to the 6C8 heavychain variable region. The amino acid sequence of the complete chimericheavy chain GITR binding molecule is shown below (also referred to a“Agly”):

(SEQ ID NO: 24) QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Example 8 Preparation of Humanized Forms of the 6C8 Anti-GITR BindingMolecule

The CDR homology based strategy described in Hwang et al. (2005) Methods(36) 35-42 was used to humanize 6C8. The heavy and light chain aminoacid sequences were blasted using a publicly available database, and theresults indicated that 6C8 had a 3-1 heavy chain canonical structure anda 2-1-1 light chain canonical structure. From this, all germ line kappachain V genes with a 2-1-1 canonical structure in the IMGT database werecompared with the 6C8 antibody sequence. The same was done for the heavychain where all 3-1 germ line heavy chain V genes were compared to the6C8 amino acid sequence. Only the CDR sequences were compared and theframeworks were selected based on which germline sequences had the mostmatches in the CDRs. (see alignments below).

For the light chain, the 3-15*01 sequence had 14 matches in the CDRs andwas selected. Since CDR 3 ends with leucine and threonine, the Jk4 Jgene segment sequence was used.

Light Chain V Genes with 2-1-1 Canonical Structure IMGT Gene Name CDR1CDR2 CDR3 IDs IGKV1-5 RASQSISSWLA...... DASSLES....... QQYNSYS.. 11IGKV1-6 RASQGIRNDLG...... AASSLSQ....... LQDYNYP.. 9 IGKV1-9RASQGISSYLA...... AASTLQS....... QQLNSYP.. 11 IGKV1-12 RASQGISSWLA......AASSLQS....... QQANSFP.. 11 IGKV1-16 RASQGISSWLA...... AASSLQS.......QQYNSYP.. 12 IGKV1D-16 RARQGISSWLA...... AASSLQS....... QQYNSYP.. 11IGKV1-17 RASQGIRNDLG...... AASSLQS....... LQHNSYP.. 9 IGKV1-27RASQGISNYLA...... AASTLQS....... QKYNSAP.. 11 IGKV1-33 QASQDISNYLN......DASNLET....... QQYDNLP.. 9 IGKV1-39 RASQSISSYLN...... AASSLQS.......QQSYSTP.. 9 IGKV1D-43 WASQGISSYLA...... YASSLQS....... QQYYSTP.. 11IGKV3-11 RASQSVSSYLA...... DASNRAT....... QQRSNWP.. 11 IGKV3D-11RASQGVSSYLA...... DASNRAT....... QQRSNWH.. 10 IGKV3-15 RASQSVSSNLA......GASTRAT....... QQYNNWP.. 14 6C8 KASQNVGTNVA...... SASYRYS....... QQYNTDP

All germ line light chain kappa chain V genes with a 2-1-1 canonicalstructure in the IMGT database were compared with the 6C8 antibodysequence. The same was done for the heavy chain where all 3-1 germ lineheavy chain V genes were compared to the 6C8 amino acid sequence

Using this methodology one version of the light chain was made:

(SEQ ID NO: 44) EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRLLIYSASYRYSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNTDPLTFGG GTKVEIK (theCDRs areitalicized)

For the heavy chain, sequence 2-05*01 had 17 matches. However, thesequences around CDR 3 were different than 6C8 (YYCAR vs. YYCAHR). SinceCDR 3 has been shown to be the most important CDR for recognition, it isimportant to keep this area as perfectly matched as possible. Sequence2-70*01 had 16 matches in the CDRs and the sequences right before CDR 3perfectly matched 6C8′s and so 2-70*01 was selected.

For the J gene segment of the heavy chain, JH4 had the most matches andwas therefore, selected. The amino acid sequences were then reversetranslated and primers corresponding to the desired nucleotide sequencewere obtained from IDT (Coralville, Iowa).

Heavy Chain V Genes with 3-1 Canonical Structures IMGT Gene Name CDR1CDR2 IDs IGHV2-5 TSGVGVG..... LIYWNDDKRYSPSLKS 17 IGHV2-26 NARMGVS.....HIFSNDEKSYSTSLKS 12 IGHV2-70 TSGMCVS..... LIDWDDDKYYSTSLKT 16 IGHV4-30-2SGGYSWS..... YIYHSGSTYYNPSLKS 10 IGHV4-30-4 SGDYYWS.....YIYYSGSTYYNPSLKS 9 IGHV4-31 SGGYYWS..... YIYYSGSTYYNPSLKS 9 IGHV4-39SSSYYWG..... SIYYSGSTYYNPSLKS 10 IGHV4-61 SGSYYWS..... YIYYSGSTNYNPSLKS8 6C8 TSGMGVG..... HIWWDDDKYYNPSLKSUsing this methodology one version of the heavy chain was made:

(SEQ ID NO: 53) QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYY

PSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCART RRYFPFAYWGQGTLVTVSS (alsoreferred to as “N”)

Since the amino acid sequence NX(S/T) is a putative consensus sequencefor a glycosylation site which may affect the production of the bindingmolecule, and CDR2 of the 6C8 heavy chain has the sequence NPS, a secondversion of the heavy chain was prepared to conservatively substitute aglutamine for an asparagine at amino acid residue 62 (bolded andunderlined above) of SEQ ID NO:53. Accordingly, a second heavy chainversion was made:

(SEQ ID NO: 54) QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYY

PSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCART RRYFPFAYWGQGTLVTVSS (alsoreferred to as “Q”).

A CLUSTAL W (1.82) multiple sequence alignment (using a Blosum scoringmatrix with a gap penalty of 10) of the 6C8 light chain variable regionand the 3-15*01 germline light chain sequence was also performed. Theresults are presented below:

6C8 DIVMTQSQKFMSTSVGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPD 3-15*01EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLIYGASTRATGIPA:******   :*.* *:*.:::*:***.*.:*:*********:*: ***.** * :*:* 6C8RFTGSGSGTDFTLTINNVHSEDLAEYFCQQYNTDPLTFGAGTKLEIK 3-15*01RFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNNWP------------ **:******:*****..::***:**:*****. *

Based on the CLUSTAL W analyses, several amino acid residues in thehuman framework were identified for potential substitution with aminoacid residues corresponding to the 6C8 framework residues in thehumanized 6C8 light chain. Specifically, the E at position 1, the P atposition 8, the A at position 9, the T at position 10, the L at position11, the V at position 13, the P at position 15, the E at position 17,the A at position 19, the T at position 20, the L at position 21, the Sat position 22, the A at position 43, the Rat position 45, the L atposition 46, the I at position 58, the A at position 60, the S atposition 63, the E at position 70, the S at position 76, the S atposition 77, the L at position 78, the Q at position 79, the F atposition 83, the V at position 85, the Y at position 87, the G atposition 100, and the V at position 104.

Similarly, a CLUSTAL W (1.82) multiple sequence alignment (using aBlosum scoring matrix with a gap penalty of 10) of the 6C8 heavy chainvariable region and the germline heavy chain proteins with a 2-70*01amino acid sequence was also performed. The results are presented below:

6C8 QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWLAHIWWDDDKY 2-70*01QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMCVSWIRQPPGKALEWLALIDWDDDKY****:****.::**:***:***:*********** *.*****.**.***** * ****** 6C8YNPSLKSQLTISKDTSRNQVFLKITSVDTADAATYYCARTRRYFPFAYWGQGTLVTVSS 2-70*01YSTSLKTRLTISKDTSKNQVVLTMTNMDPVDTATYYCARI-------------------*..***::********:***.*.:*.:*..*:*******

Based on the CLUSTAL W analyses, several amino acid residues in thehuman framework were identified for potential substitution with aminoacid residues corresponding to the 6C8 framework residues in thehumanized 6C8 heavy chain.

Specifically, the R at position 5, the A at position 10, the L atposition 11, the V at position 12, the T at positionl5, the T atposition 19, the T at position 23, the P at position 43, the A atposition 46, the R at position 68, the K at position77, the V atposition 81, the T at position 83, the M at position 84, the N atposition 86, the M at position 87, the P at position 89, the V atposition 90, and/or the T at position 92.

Four humanized full-length 6C8 binding molecules were made having thefollowing humanized heavy and light chain combinations:

Full-length Version 1 (HuN6C8-Gly)—humanized (Hu) 6C8 Light chain(L)/humanized Heavy chain with the N in CDR2 (“N”) and comprising aconstant region having an N (“Gly”) Full-length Version 2(HuN6C8-Agly)—humanized (Hu) 6C8 Light chain (L)/humanized Heavy chainwith the N in CDR2 (“N”) and comprising a constant region having an A(“Agly”) Full-length Version 3—(HuQ6C8-Gly)—humanized (Hu) 6C8 Lightchain (L)/humanized Heavy chain with the Q in CDR2 (“Q”) and comprisinga constant region having an N (“Gly”) Full-length Version4—(HuQ6C8-Agly)—humanized (Hu) 6C8 Light chain (L)/humanized Heavy chainwith the Q in CDR2 (“Q”) and comprising a constant region having an A(“Agly”)

The amino acid sequence of the glycosylated IgG1 heavy chain constantregion that was used to make the full-length binding molecules is shownbelow:

(SEQ ID NO: 55) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

The amino acid sequence of the aglycosylated IgG1 heavy chain constantregion that was used to make the full-length binding molecules is shownbelow:

(SEQ ID NO: 56) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

The amino acid sequence of the IgG1 light chain constant region that wasused to make the full-length binding molecules is shown below:

(SEQ ID NO: 57) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC.

The complete amino acid sequence of the humanized 6C8 light chain isshown below:

(SEQ ID NO: 58) EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRLLIYSASYRYSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNTDPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.The leader sequence METQSQVFVYMLLWLSGVDG (SEQ ID NO:59) may optionallybe included.

The complete amino acid sequences of the humanized 6C8 heavy chainversions HuN6C8-Agly, HuQ6C8-Gly, and HuQ6C8-Agly are shown below:

HuN6C8-Gly (SEQ ID NO: 60)QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYNPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; HuN6C8-Agly (SEQ IDNO: 61) QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYNPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; HuQ6C8-Gly (SEQ IDNO: 62) QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYQPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK; and HuQ6C8-Agly (SEQID NO: 63) QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMGVGWIRQPPGKALEWLAHIWWDDDKYYQPSLKSRLTISKDTSKNQVVLTMTNMDPVDTATYYCARTRRYFPFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.The leader sequence MDRLTFSFLLLIVPAYVLS (SEQ ID NO:64) may optionally beincluded.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1-57. (canceled)
 58. A method for inducing or enhancing an immuneresponse in a subject, the method comprising: administering to thesubject an agonistic GITR (glucocorticoid-induced TNFR family-relatedreceptor)-binding antibody, or an antigen-binding fragment thereof, andan antigen, such that an immune response or an enhanced immune responseoccurs, wherein the antibody or antigen-binding fragment thereofcomprises the light chain complementarity determining region (CDR) aminoacid sequences shown in amino acid residues 44-54 of SEQ ID NO:2, aminoacid residues 70-76 of SEQ ID NO:2, and amino acid residues 109-117 ofSEQ ID NO:2, and heavy chain CDR amino acid sequences shown in aminoacid residues 45-56 of SEQ ID NO:1, amino acid residues 119-127 of SEQID NO:1, and one of amino acid residues 71-86 of SEQ ID NO:1 and aminoacid residues 71-86 of SEQ ID NO:66; and administering to the subject anadditional antibody or antigen-binding fragment thereof or giving thesubject a treatment selected from the group consisting of chemotherapyand hormonal therapy.
 59. The method of claim 58, wherein the subjectcomprises a source of antigen to which the immune response is directed.60. The method of claim 59, wherein the source of antigen comprises atumor or an infectious microorganism.
 61. The method of claim 58,wherein the immune response comprises a humoral immune response.
 62. Themethod of claim 58, wherein binding of the GITR-binding antibody or theantigen-binding fragment to a T cell results in abrogation ofsuppression of a T-effector cell by a T-regulatory cell.
 63. The methodof claim 58, wherein the GITR-binding antibody or the antigen bindingfragment induces or enhances proliferation of a T-effector cell.
 64. Themethod of claim 58, wherein binding of the GITR-binding antibody or theantigen-binding fragment to a T-effector cell results in induction orenhancement of proliferation of the T-effector cell.
 65. The method ofclaim 58, wherein binding of the GITR-binding antibody or theantigen-binding fragment to a T cell results in modulation of I-κB inthe T cell.
 66. The method of claim 58, wherein binding of theGITR-binding antibody or the antigen-binding fragment to a T cellresults in modulation of GITR activity in the T cell.
 67. The method ofclaim 58, wherein binding of the GITR-binding antibody orantigen-binding fragment to a T cell results in T cell receptor-inducedsignaling in a T-effector cell.
 68. A method for inducing or enhancingan immune response to an antigen in a subject, the method comprising:administering to the subject an agonistic GITR (glucocorticoid-inducedTNFR family-related receptor)-binding antibody, or an antigen-bindingfragment thereof, and an antigen, such that an immune response or anenhanced immune response occurs, wherein the antibody or antigen-bindingfragment thereof comprises the light chain complementarity determiningregion (CDR) amino acid sequences shown in amino acid residues 44-54 ofSEQ ID NO:2, amino acid residues 70-76 of SEQ ID NO:2, and amino acidresidues 109-117 of SEQ ID NO:2, and heavy chain CDR amino acidsequences shown in amino acid residues 45-56 of SEQ ID NO:1, amino acidresidues 119-127 of SEQ ID NO:1, and one of amino acid residues 71-86 ofSEQ ID NO:1 and amino acid residues 71-86 of SEQ ID NO:66; andadministering to the subject an additional antibody or antigen-bindingfragment thereof or giving the subject a treatment selected from thegroup consisting of chemotherapy and hormonal therapy.
 69. The method ofclaim 68, wherein the antigen is a tumor antigen or an infectiousmicroorganism antigen.
 70. The method of claim 68, wherein the immuneresponse comprises a humoral immune response.
 71. The method of claim68, wherein binding of the GITR-binding antibody or the antigen-bindingfragment to a T cell results in abrogation of suppression of aT-effector cell by a T-regulatory cell.
 72. The method of claim 68,wherein administration of the GITR-binding antibody or theantigen-binding fragment induces or enhances proliferation of aT-effector cell.
 73. The method of claim 68, wherein binding of theGITR-binding antibody or the antigen-binding fragment to a T-effectorcell results in induction or enhancement of proliferation of theT-effector cell.
 74. The method of claim 68, wherein binding of theGITR-binding antibody or the antigen-binding fragment to a T cellresults in modulation of I-κB in the T cell.
 75. The method of claim 68,wherein binding of the GITR-binding antibody or the antigen-bindingfragment to a T cell results in modulation of GITR activity in the Tcell.
 76. The method of claim 68, wherein binding of the GITR-bindingantibody or antigen-binding fragment to a T cell results in T cellreceptor-induced signaling in a T-effector cell.
 77. The method of claim68, wherein the antigen is administered at least one time prior to theadministration of, or is co-administered at least one time with, theGITR-binding antibody or the antigen-binding fragment.
 78. The method ofclaim 58, wherein the antibody or antigen-binding fragment thereofcomprises a humanized light chain amino acid sequence set forth in SEQID NO: 58 and a humanized heavy chain amino acid sequence set forth inSEQ ID NO:
 63. 79. The method of claim 68, wherein the antibody orantigen-binding fragment thereof comprises a humanized light chain aminoacid sequence set forth in SEQ ID NO: 58 and a humanized heavy chainamino acid sequence set forth in SEQ ID NO: 63.